Sugar chain-containing water-soluble polymer compound

ABSTRACT

A polymer compound having a monosaccharide or an oligosaccharide residue, or an amino acid or peptide residue bound to a monosaccharide or an oligosaccharide residue bound to a side chain of a water-soluble polymer through a linker containing a selectively cleavable bond, the water-soluble polymer containing 20 to 80 mol % of (meth)acrylic acid residue.

TECHNICAL FIELD

The present invention relates to a water-soluble polymer compound havingsugar chains that is useful for producing glycocunjugates, a method forproducing the compound, a method for producing glycoconjugates by usingthe compound, and a water-soluble polymer primer comprising the compoundfor synthesizing glycoconjugates.

BACKGROUND OF THE INVENTION

Although carbohydrates are just as important constituents of livingbodies as nucleic acids and proteins, their structures and mechanismsare not as well understood as those of nucleic acids and proteins.Carbohydrates frequently form polymers having sugar chain in sequence,and bind with proteins or lipids to form extremely complicated compositemolecules, glycoconjugates, which are called glycoproteins, glycolipids,and proteoglycans. Nucleic acids and proteins are polymers whereinconstituent units of nucleotides or amino acids are linked to one otherlinearly. In contrast, carbohydrates have a plurality of intramolecularbranch points and their constitutional units, i.e., monosaccharides, arelinked to each other in various manners, and therefore carbohydrateshave complicated structures incomparable to those of nucleic acids orproteins. This structural complexity is one of the major causes of thedelay in the study of carbohydrates.

In recent years, since it has been gradually revealed that carbohydrateshave a roll in cell recognition, immunity, differentiation,fertilization, aging, canceration, etc., carbohydrates become a targetof study that attracts significant attention. Under such circumstances,many attempts have been made to synthesize a sugar chain having naturalstructure and a novel sugar chain. Automatic synthesis techniques fornucleic acids and proteins have been already established, and thesetechniques have obviously accelerated the progress of the researches inthese fields. Therefore, the establishment of automatic synthesistechniques for sugar chains have been eagerly desired.

Several attempt for automatic synthesis for sugar chains have so farbeen reported, and their approaches can be roughly classified into twogroups. One employs chemical synthesis, which has many problems such asthe fact that stereoselective glycosylation reaction has not been wellestablished, and the process is tedious and complicate because ofprotection and deprotection. Another employs enzymatic synthesis, whichrequires no protection, and glycosylation reaction can be carried outstereoselectively. Therefore, compared to chemical synthesis, enzymaticsynthesis has many advantages. Several methods have been proposed inrecent years. It is due to cloning the genes of variousglycosyltransferases and economical production of some recombinantglycosyltransferases. In automatic synthesis, a certain carrier(sometimes referred to as a primer) bound sugar residues, which serve asinitiators, through linkers that can be cleaved under specificconditions is used as a starting material. The types of glycoconjugateproduced depend on the kind of linkers, and they are released from thecarrier as oligosaccharides, glucosides, glycopeptides, glycolipids,etc.

As one of the examples of automatic synthesis method of a sugar chainusing glycosyltransferases, U. Zehavi et al. have reported on asolid-phase synthesis method using a polyacrylamide gel bound with anaminoethyl group or an aminohexyl group as a solid-phase carrier (see,for example, Carbohydr. Res., 124(1983), 23; and Carbohydr. Res.,228(1992), 255). This method comprises the steps of converting asuitable monosaccharide to 4-carboxy-2-nitrobenzyl glycoside, condensingthis glycoside with amino group of the above carrier, elongating thesugar chain by glycosyltransferase using the condensate as a primer, andreleasing the elongated sugar chain as oligosaccharide by photolysis.According to this method, however, sugar transfer yield is low, i.e.,less than 10%. It has been a common understanding thatglycosyltransferase dose not react well with monosaccharide oroligosaccharide bound to a solid-phase carrier and efficient elongationof sugar chain reaction is difficult to achieve. However, U. Zehavi etal. have documented in a recent report that the sugar transfer yieldcould be improved up to 51% by using a linker between4-carboxy-2-nitrobenzyl glycoside and the solid-phase carrier having along chain such as hexamethylene, and octamethylene, etc. (see, forexample, React. Polym., 22(1994), 171; Carbohydr. Res., 265(1994), 161).However, even this method cannot achieve satisfactory yields.

As another example, C.-H. Wong et al. have reported a method wherein asugar chain is elongated using glycosyltransferases and aminated silicabound a group represented by the following formula

(wherein Ac is an acetyl group and Boc is a t-butoxycarbonyl group) as aprimer, and the elongated sugar chain was released in the form of aglycopeptide by hydrolysis of α-chymotrypsin (see, for example, J. Am.Chem. Soc., 116(1994), 1136). However, the yield of sugar-chainelongation reaction using glycosyltransferase is unsatisfactory at 55 to65%.

Furthermore, C.-H. Wong et al. revised the group to be bound to thesolid phase as aminated silica to represented by the following formula

(wherein Ac is an acetyl group) and reported a method wherein the sugarchain was elongated using glycosyltransferases and released byhydrazinolysis. They also reported that the enzymatic glycosylationreaction was proceeded almost quantitatively (see, for example, J. Am.Chem. Soc., 116(1994), 11315). In this method, the elongated sugar chainis released in the form of a 6-carbohydrazide hexanol glucoside.

M. Meldal et al. have reported a method wherein a sugar chain waselongated using glycosyltransferases and a polymer gel of mono- anddiacryloyl compound of diaminated poly(ethylene glycol) having a grouprepresented by the following formula

(wherein Ac is an acetyl group) as a primer and the elongated sugarchain was released in the form of a glycopeptide using trifluoroaceticacid. According to their report, the transglycosylation reaction isproceeded almost quantitatively (see, for example, J. Chem. Soc., Chem.Commun., 1849 (1994)). The peptide sequence in the glycopeptide obtainedby this method is Asn (asparagine)-Gly (glycine), and the glycineresidue at the C-end is a glycinamide residue, and therefore it differsfrom typical glycopeptides. C.-H. Wong et al. have also reported amethod for releasing typical glycopeptides synthesized on a solid-phasecarrier. In this method, aminated silica is used as a solid-phasecarrier introduced a group represented by the following formula as aprimer

(wherein Fmoc is (9-fluorenylmethyl)oxycarbonyl)), the peptide chain iselongated using Fmoc-amino acids and Fmoc-Thr(βGlcNAc)-OH, after peptideelongation protecting groups on the peptide chain are eliminated, thesugar chain is elongated by glycosyltransferase to the above-mentionedN-acetylglucosamine residue, and resultant glycopeptide is released bytetrakis(triphenylphosphine)palladium treatment (see, for example, J.Am. Chem. Soc., 119(1997), 8766). The yield of the obtained glycopeptideestimated from the amino acid initially introduced to the solid-phasecarrier is less that 10%, which is unsatisfactory.

T. Norberg et al. have reported a method wherein a sugar chain waselongated using Sepharose 6B (manufactured by Amersham PharmaciaBiotech) bound a group represented by the following formula as a primerand glycosyltransferase,

and the elongated sugar chain is released by treatment with bromine orammonia/ammonium borate (see, for example, Carbohydr. Res., 319(1999),80). In this method, the enzymatic transglycosylation reaction proceedsquantitatively, so there is no problem with the yield. However, thismethod is uneconomical because expensive3,4-diethoxy-3-cyclobutene-1,2-dione is used for producing the primer.The above-described methods have drawbacks such that thetransglycosylation reaction yield is unsatisfactory, and/or animmobilized glycosyltransferase cannot be appiled since the sugar-chainelongation reaction is carried out on a water-insoluble carrier. Insugar chain elongation by glycosyltransferases, the use of immobilizedglycosyltransferases that permit repetitive use are desirable, sinceglycosyltransferases are still very expensive, though mass production ofglycosyltransferases by genetic recombination techniques are becomingavailable. In order to use an immobilized glycosyltransferase, thesugar-chain elongation reaction should be proceeded not on awater-insoluble carrier but on a water-soluble carrier.

S. Roth et al. have disclosed a method as follows (see, for example,Japanese Unexamined Patent Publication No. 1993-500905). Saccharide,sugar acceptor for a glycosyltransferase, is bound to a solid-phasecarrier to form an affinity adsorbent, and a glycosyltransferase is thenadsorbed to the above adsorbent by contacting with a tissue extractcontaining a glycosyltransferase that can recognize the sugar acceptor.Thereafter, the glycosyltransferase transfers sugar residue from sugarnucleotide to the acceptor on the absorbent and elutes from theadsorbent by contacting with a solution containing a sugar nucleotidewhich the glycosyltransferase can use as a sugar donor. Furthermore, byrepetition of contacting the resultant one sugar residue elongated sugarchain on the absorbent with a tissue extract containing anotherglycosyltransferase that can recognize the elongated sugar acceptor andthe similar elution procedure, a desired sugar chain can be synthesizedon a solid-phase carrier. However, no concrete data demonstrating theeffectiveness of this method is provided. Furthermore, no methods forreleasing the elongated sugar chain from the solid-phase carrier aredisclosed.

C.-H. Wong et al. have also reported a method for elongating a sugarchain on a water-soluble carrier wherein a water-soluble polymer,acrylamide/acrylic acid/N-isopropylacrylamide copolymer bound a grouprepresented by the following formula to acrylamide residue in thiscopolymer

(wherein Ac is an acetyl group) is used as a primer, the sugar chain iselongated by glycosyltransferases and released by the treatment withcerium (IV) diammonium nitrate (see, for example, Adv. Synth. Catal.,343(2001), 675). The proportion of acrylic acid in the copolymer primerused in this method is 4%, and therefore this primer differs from thatof the present invention. In this method, the enzymatictransglycosylation reaction progresses at 80 to 90% yield, and theelongated sugar chain is released in the form of a p-formylphenolglucoside. However, this method has drawbacks such that columnchromatography using organic solvents is required to purify the releasedp-formylphenol glucoside, and in some cases obtained p-formylphenolglucoside is not so stable.

The present inventors have reported a method for elongating a sugarchain on a water-soluble carrier wherein polyacrylamide bound a grouprepresented by the following formula

(wherein Ac is an acetyl group) to every fifth amide nitrogen atom in itis used as a primer, the sugar chain is elongated byglycosyltransferases, and the elongated sugar chain is released in theform of an oligosaccharide by hydrogenolysis (see, for example,Tetrahedron Lett., 0.35(1994), 5657; Carbohydr. Res., 305(1998), 443).

The present inventors have also reported a method for elongating a sugarchain on a water-soluble carrier wherein polyacrylamide bound a grouprepresented by the following formula

(wherein Ac is an acetyl group) to an amide nitrogen atom of amidemoiety is used as a primer, the sugar chain is elongatedglycosyltransferases, and the elongated sugar chain is released in theform of a 6-aminohexanol glucoside by hydrolysis of α-chymotrypsin (see,for example, Tetrahedron Lett., 36(1995), 9493; Carbohydr. Res.,305(1998), 443).

According to these reports by the present inventors, glycoconjugates canbe efficiently synthesized by free enzymes; however, as described later,when an immobilized enzyme is used, the production efficiency isunsatisfactory. The present inventors have also reported a method forelongating a sugar chain on a water-soluble carrier whereinpolyacrylamide bound peptide residue linked monosaccharide residue tofunctional group on side chain of amino acid in this peptide residuethrough a linker which has a desired length and comprises an amino acidresidue or peptide residue having a cleavage site for a certain proteasefor example a group represented by the following formula

(wherein Ac is an acetyl group) is used as a primer, a certain proteasedose not have cleavable site in the above-mentioned peptide which bounddirectly polyacrylamide, the sugar chain elongation is initiated on theabove-mentioned monosaccharide by glycosyltransferases, and theelongated sugar chain is released in the form of a glycopeptide using aappropriate protease hydrolysis (see, for example, Japanese UnexaminedPatent Publication No. 2001-220399).

The present inventors have also reported a method which uses a primercomprising a residue represented by the following formula (VIII)

(wherein R¹³ and R¹⁴ are independently H, a monosaccharide residue or anoligosaccharide residue; R¹⁵ is a C₆₋₂₀ alkyl group or C₆₋₂₀ alkenylgroup; and R¹⁶ is a C₅₋₁₉ alkylene group) bound to a side chain of awater-soluble polymer. In this method, the sugar chain is elongated byglycosyltransferases, an resultant oligosaccharide residue istransferred in the presence of a ceramid from the polymer havingelongated sugar chain to the ceramide by ceramide glycanase, and thenliberated as a sphingoglycolipid (see, for example, Japanese UnexaminedPatent Publication No. 1998-251287).

In the above-described methods, polyacrylamide is disclosed as oneexample of water-soluble polymer but no examples are disclosed whenacrylic acid is used. Furthermore, when a polyacrylamide is used as awater-soluble polymer, if the transglycosylation reaction is proceededby immobilized glycosyltransferases, its efficiency is unsatisfactory asdescribed later. If the latter primer is used, when gel filtrationchromatography and ultrafiltration are performed to remove by-productnucleotides and unreacted sugar nucleotides after the transglycosylationreaction, recovery of the primer is not always satisfactorily high.

One of the main objects of the present invention is to provide acompound that is suitable and useful as a primer for automatic synthesisof various kinds of glicoconjugates, and a method for producingglycoconjugate using the compound.

DISCLOSURE OF THE INVENTION

The present inventors conducted extensive research and found that theabove problems are solved by using a water-soluble polymer as primerwhich contains 20 to 80 mol % of (meth)acrylic acid-based residues(selected from the group consisting of acrylic acid and salts thereof,and methacrylic acid and salts thereof) and is bound a monosaccharideresidue, an oligosaccharide residue, or a peptide to which amonosaccharide residue or an oligosaccharide residue to a side chain ofthis polymer through a linker containing a selectively cleavable bond.The present invention has been accomplished based on this finding.

In other words, the present invention encompasses the following items.

1. A water-soluble polymer compound having sugar chain(s) comprising amonosaccharide or an oligosaccharide residue bound to side chain(s) of awater-soluble polymer through a linker containing a selectivelycleavable bond, the water-soluble polymer containing 20 to 80 mol % of(meth)acrylic acid residue, and the linker being bonded to a repeatingunit other than (meth)acrylic acid residue.

2. A compound according to Item 1, wherein amino acid or peptideresidues bound to a monosaccharide or an oligosaccharide residue arelinked to side chain(s) of the water-soluble polymer through a linkercontaining a selectively cleavable bond, the water-soluble polymercontaining 20 to 80 mol % of (meth)acrylic acid residue, and the linkerbeing bound to a repeating unit other than (meth)acrylic acid residue.

3. A compound according to Item 1 or 2, wherein the water-solublepolymer is a copolymer comprising 20 to 80 mol % of (meth)acrylic acidand 80 to 20 mol % of one or more vinyl monomers selected from the groupconsisting of acrylamide derivatives, methacrylamide derivatives,acrylic esters, methacrylic esters, styrene derivatives and fatty-acidvinyl esters.

4. A compound according to any one of Items 1 to 3, wherein theselectively cleavable bond contained in the linker can be cleaved byhydrogenolysis or by oxidation using2,3-dichloro-5,6-dicyanobenzoquinone.

5. A compound according to any one of Items 1 to 4, wherein the linkeris a group represented by General Formula (I),

wherein R¹ is a monosaccharide or an oligosaccharide residue, R² is abivalent linking group with a length equivalent to 4 to 20 methylenegroups, and X is O, S, or NH.

6. A compound according to Item 5, wherein R¹ is an N-acetylglucosamineresidue, a glucose residue or a lactose residue.

7. A compound according to Item 5 or 6, wherein R² is a pentylene group.

8. A compound according to any one of Items 1 to 7, wherein the linkeris a group represented by General Formula (II),

wherein R³ is a monosaccharide or an oligosaccharide residue, R⁴ is aC₆₋₂₀ alkyl or alkenyl group, R⁵ is a bivalent linking group with alength equivalent to 5 to 19 methylene groups, and Y is O, S, or NH.

9. A compound according to Item 8, wherein R³ is a glucose or lactoseresidue.

10. A compound according to any one of Items 1 to 9, wherein the linkeris a group represented by General Formula (III),

wherein R⁶ is a monosaccharide or an oligosaccharide residue, R⁷ is abivalent linking group with a length equivalent to 2 to 20 methylenegroups, R⁸ is a bivalent linking group with a length equivalent to 5 to19 methylene groups, and Z and W are each independently O, S, or NH.

11. A compound according to Item 10, wherein R⁶ is anN-acetylglucosamine residue.

12. A compound according to Item 2, wherein the peptide residue consistsof 2 to 30 amino acid residues.

13. A compound according to any one of Items 1 to 12, wherein theselectively cleavable bond contained in the linker can be cleaved by anappropriate hydrolase.

14. A compound according to Item 13, wherein the appropriate hydrolaseis ceramide glycanase or α-chymotrypsin.

15. A compound according to Item 13, wherein the appropriate hydrolaseis a protease that does not have a cleavage site in amino acid orpeptide residue to which a monosaccharide or an oligosaccharide residueis bound.

16. A compound according to Item 15, wherein the linker containing aselectively cleavable bond that is linked to an amino acid or a peptideresidue bound to a monosaccharide or an oligosaccharide residue is agroup represented by General Formula (IV),—R⁹-R¹⁰—  (IV)wherein R⁹ is a bivalent linking group with a length equivalent to 1 to20 methylene groups and is linked to the water-soluble polymer compound,and R¹⁰ is an amino acid or a peptide residue containing a cleavablesite by an appropriate protease and is bound to a monosaccharide or anoligosaccharide residue, and that the monosaccharide or oligosaccharideresidue is bound to a side chain functional group of Asn, Asp, Cys, Gln,Glu, Lys, Ser, Thr or Tyr residue, or to a side chain functional groupof the amino acid residue in a peptide residue directly or through abivalent linking group via a glycosidic bond.

17. A compound according to Item 16, wherein R⁹ is a group representedby General Formula (V),-A-(CH₂)_(n)—CO—  (V)wherein A is O, CH₂, C═O, or NH, the group is linked to a side chain ofthe water-soluble polymer through A, and n is an integer from 1 to 18.

18. A compound according to Item 16 or 17, wherein the bivalent linkinggroup bound to the side chain functional group is a group with a lengthequivalent to 1 to 20 methylene groups.

19. A compound according to any one of Items 16 to 18, wherein thebivalent linking group linked to the side chain functional group is agroup represented by General Formula (VI),—B—(CH₂)_(n)—O—  (VI)wherein B is O, NH, or C═O, the group is linked to the side chainfunctional group of an amino acid residue through B, and n is an integerfrom 1 to 18.

20. A water-soluble polymer primer for glycoconjugate synthesiscomprising a water-soluble polymer compound having sugar chain(s)according to any one of Items 1 to 19.

21. A method for producing a water-soluble polymer compound having sugarchain(s) comprising a step of copolymerization of (meth)acrylic acid, a(meth)acrylamide derivative represented by General Formula (VII),

wherein R¹¹ is a monosaccharide or an oligosaccharide residue, and R¹²is a bivalent linking group with a length equivalent to 4 to 20methylene groups, and at least one vinyl monomer in such a manner thatthe proportion of the (meth)acrylic acid in the total vinyl copolymersis 20 to 80 mol %.

22. A method according to Item 21, wherein R¹¹ is an N-acetylglucosamineresidue, a glucose residue, or a lactose residue.

23. A method according to Item 21, wherein R¹² is a pentylene group.

24. A method according to Item 21, wherein the vinyl monomer is at leastone monomer selected from the group consisting of acrylamidederivatives, methacrylamide derivatives, acrylic esters, methacrylicacid esters, styrene derivatives, and fatty acid vinyl esters.

25. A method for producing a glycoconjugate comprising the steps of:

(step 1) transferring a sugar residue from a sugar nucleotide to apolymer compound by contacting a water-soluble polymer compound havingsugar chain(s) of Item 1 or 2 with a glycosyltransferase in the presenceof a sugar nucleotide,

(step 2) elongating the sugar chain by repeating step 1 two or moretimes if necessary,

(step 3) removing by-product nucleotides or unreacted sugar nucleotidesif necessary, and

(step 4) after repeating steps 1 to 3 two or more times, releasing theresultant glycoconjugate sugar chain from the water-soluble polymercompound which binds the sugar chain elongated by the transfer of theplurality of sugar residues.

26. A method for producing a glycoconjugate compound comprising thesteps of:

(step 1) transferring a sugar residue from a sugar nucleotide to awater-soluble polymer compound by the action of a glycosyltransferase tothe water-soluble polymer compound having sugar chain(s) of Item 8 inthe presence of a sugar nucleotide,

(step 2) elongating the sugar chain by repeating step 1 two or moretimes if necessary,

(step 3) removing by-product nucleotides or unreacted sugar nucleotidesif necessary and

(step 4) after repeating steps 1 to 3 two or more times, transferringthe resultant oligosaccharide elongated by transfer of the plurality ofsugar residues from the water-soluble polymer compound to ceramide bythe action of ceramide glycanase in the presence of ceramide.

Various glycoconjugates can be efficiently synthesized by using thesugar chain-having water-soluble polymer compound of the presentinvention as a water-soluble polymer primer for glycoconjugate synthesis(which hereunder may be simply referred to as a “primer”), and theprimer can be also used for automatic synthesis of glycoconjugates.

The present invention is explained in detail below.

The water-soluble polymer compound having sugar chain(s) of the presentinvention (which hereunder may be referred to as the “sugar chain-havingpolymer”) is a polymer in which monosaccharide residues oroligosaccharide residues are bound to side chain(s) of a water-solublepolymer through a linker containing a selectively cleavable bond, withthe water-soluble polymer usually containing 20 to 80 mol %, preferably30 to 70 mol %, and more preferably 40 to 60 mol % of (meth)acrylic acidresidues as monomers.

In other words, the water-soluble polymer of the present inventioncontains 20 to 80 mol % of (meth)acrylic acid residues (includingcarboxyl group salts) represented by the following formulae:

wherein M is a hydrogen atom, an alkali metal (Na, Li, K), ½ an alkalineearth metal (½Ca, ½Mg, ½Ba), ammonium, etc.

Here, the sugar chain is bound to a repeating unit derived from amonomer other than (meth)acrylic acid residue, such as a repeating unithaving one of the following structures:

In one preferable embodiment, a linker is linked to a repeating unitderived from acrylic acid or methacrylic acid via an ester linkage(—COO—), an amide linkage (—CONH—), or a thioester linkage (—COS—), andthe linker is bonded to a monosaccharide or an oligosaccharide residuethrough a glycosidic bond.

Examples of monosaccharides include glucose, galactose, mannose, xylose,N-acetylglucosamine, N-acetylgalactosamine, but are not limited tothese.

Examples of oligosaccharides include those comprising 2 to 10 of theabove-mentioned monosaccharides linked to one another, such as lactose,chitobiose, N-acetyllactosamine, α-2,3-sialyllactose,3-β-galactosyl-(6-β-N-acetylglucosaminyl)-N-acetylgalactosamine, etc.The oligosaccharide may have a linear structure or have a branchedstructure in which one sugar residue is bound to two sugar residues.

The sugar chain-having polymer of the present invention is a polymer inwhich amino acid or peptide residues bound to a monosaccharide residueor oligosaccharide residue are linked to side chains of thewater-soluble polymer through a linker containing a selectivelycleavable bond. The sugar chain-having polymer contains 20 to 80 mol %of (meth)acrylic acid residues as monomers. The polymer may have apartial structure as below:

A sugar chain-having polymer whose sugar chain has been elongated byapplying the action of a glycosyltransferase is also encompassed in thepolymer of the invention if the sugar chain thereof can be furtherelongated by a glycosyltransferase.

The monomers of the water-soluble polymer other than (meth)acrylic acid(20 to 80 mol %) are not limited and polymers of at least one vinylmonomer selected from the group consisting of acrylamide derivatives,methacrylamide derivatives, acrylic esters, methacrylic esters, styrenederivatives, and fatty acid vinyl esters can be suitably used. Amongthese, sugar chains of monosaccharides, oligosaccharides, etc., can bebound preferably to acrylamide derivatives, methacrylamide derivatives,acrylic esters, and methacrylic esters through a linker or, ifnecessary, an amino acid residue or a peptide residue.

Note that acrylamide derivatives, methacrylamide derivatives,hydroxyethyl acrylate and like hydroxyalkyl esters of acrylic acid;dimethylaminoethyl acrylate and like dimethylaminoalkyl esters ofacrylic acid; hydroxyethyl methacrylate and like hydroxyalkyl esters ofmethacrylic acid; dimethylaminoethyl methacrylate and likedimethylaminoalkyl esters are highly water soluble and may be used in alarge amount (for example, 70 mol % or more). However, other acrylicesters, other methacrylic esters, styrene derivatives, and fatty acidvinyl esters can be used only in an amount such that the polymer as awhole is water soluble. Acrylamide derivatives, methacrylamidederivatives, hydroxyethyl acrylate and like hydroxyalkyl esters ofacrylic acid; dimethylaminoethyl acrylate and like dimethylaminoalkylesters of acrylic acid; hydroxyethyl methacrylate and likemethhydroxyalkyl esters of acrylic acid; dimethylaminoethyl acrylate andlike dimethylaminoalkyl esters may be used in an amount calculated bysubtracting the amount of (meth)acrylic acid (20 to 80 mol %) and thatof the sugar chain-having monomers.

Acrylamide and N-alkylacrylamides such as N-methylacrylamide,N-ethylacrylamide, N-isopropylacrylamide and the like are preferablyused as acrylamide derivatives.

Methacrylamide and N-alkylmethacrylamides such asN-methylmethacrylamide, N-isopropylmethacrylamide and the like arepreferably used as methacrylamide derivatives.

Methyl acrylate, ethyl acrylate, hydroxyethyl acrylate,dimethylaminoethyl acrylate and the like are preferably used as theacrylic esters.

Methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate,dimethylaminoethyl methacrylate are preferably used as methacrylicesters.

Styrene, p-hydroxystyrene and the like are preferably used as styrenes.

Vinyl acetate, vinyl butyrate and the like are preferably used as fattyacid vinyl esters.

Selectively cleavable bonds are not limited as long as the sugar chaincompound released after cleavage, such as an oligosaccharide,glycopeptide, sphingoglycolipid, oligosaccharide glucoside, is cleavedwithout being decomposed, and it is possible that several bonds includedin linkers used in solid phase synthesis of peptides or oligonucleotidescan be used. For example, bonds cleavable by weak acid or weak alkali,bonds cleavable by hydrogenolysis, bonds cleavable by photoreaction,bonds cleavable by enzymatic reaction, etc., can be used. Morepreferable examples thereof include hydrogenolysis, oxidation using2,3-dichloro-5,6-dicyanobenzoquinone, hydrolysis using a protease,transglycosylation of ceramide glycanase, etc.

There is no limitation to monosaccharide or oligosaccharide residuebound to a side chain of the water-soluble polymer through a linkerhaving a selectively cleavable bond. Examples thereof include groupsrepresented by General Formula (I) (wherein R¹ is a monosaccharide or anoligosaccharide residue, R² is a bivalent linking group with a lengthequivalent to 4 to 20 methylene groups, and X is O, S, or NH), groupsrepresented by General Formula (II) (wherein R³ is a monosaccharide oran oligosaccharide residue, R⁴ is a C₆₋₂₀ alkyl or alkenyl group, R⁵ isa bivalent linking group with a length equivalent to 5 to 19 methylenegroups, and Y is O, S, or NH), groups represented by General Formula(III) (wherein R⁶ is a monosaccharide or an oligosaccharide residue, R⁷is a bivalent linking group with a length equivalent to 2 to 20methylene groups, R⁸ is a bivalent linking group with a lengthequivalent to 5 to 19 methylene groups, and Z and W are independently O,S, or NH), etc.

In General Formula (I), all the part except for R¹—O— is the linkerhaving a selectively cleavable bond, in General Formula (II), all thepart except for R³—O— is the linker having a selectively cleavable bond,and in General Formula (III) all the part except for R⁶—O— is the linkerhaving a selectively cleavable bond.

Here, such a bivalent linking group with a length equivalent to 4 to 20methylene groups (preferably a length equivalent to 4 to 18 methylenegroups, and more preferably a length equivalent to 6 to 12 methylenegroups), a bivalent linking group with a length equivalent to 5 to 19methylene groups (preferably a bivalent linking group with a lengthequivalent to 5 to 15 methylene groups, and more preferably a bivalentlinking group with a length equivalent to 5 to 11 methylene groups), anda bivalent linking group with a length equivalent to 2 to 20 methylenegroups (preferably a bivalent linking group with a length equivalent to4 to 18 methylene groups, and more preferably a bivalent linking groupwith a length equivalent to 6 to 12 methylene groups) may consist ofmethylene residues, or a group in which methylene residues are linked byether linkages (for example, —(OCH₂CH₂)n₁— wherein n₁ is an integer from1 to 6; however, the length as the whole is equivalent to 4 to 20methylene groups, 5 to 19 methylene groups, or 2 to 20 methylene groups,with 0 being equivalent to one methylene group). The same applies toother bivalent linking groups in the present specification.

The monosaccharide residue or oligosaccharide residue of R¹ is notlimited, and examples thereof include glucose residue, galactoseresidue, mannose residue, N-acetylglucosamine residue,N-acetylgalactosamine residue, xylose residue, lactose residue,N-acetyllactosamine residue, chitobiose residue, etc.

There is no limitation to a bivalent linking group of R² with a lengthequivalent to 4 to 20 methylene groups, and, for example, C₄₋₂₀ alkylenegroups may be used. Examples of C₄₋₂₀ alkylene groups include butylenesgroup, pentylene group, heptylene group, dodecylene group, etc.

The monosaccharide or oligosaccharide residue of R³ is not limited, andexamples thereof include glucose residue, lactose residue, etc.Specifically preferable examples thereof include β-glucose residue andβ-lactose residue.

There is no limitation to the C₆₋₂₀ alkyl group of R⁴, and hexyl, octyl,dodecyl, octadecyl groups and like linear or branched C₆₋₂₀ (preferablyC₈₋₁₈) alkyl groups can be used. There is no limitation to C₆₋₂₀ alkenylgroups and examples thereof include cis-9-octadecenyl and like linear orbranched C₆₋₂₀ (preferably C₈₋₁₈) alkenyl groups.

There is no limitation to the bivalent linking group of R⁵ with a lengthequivalent to 5 to 19 methylene groups, and examples thereof includeC₅₋₁₉ alkylene groups, etc. Examples of C₅₋₁₉ alkylene groups includepentylene group, heptylene group, nonylene group, heptadecylene group,etc.

There is no limitation to the monosaccharide residue or oligosaccharideresidue of R⁶, and examples thereof include glucose residue, galactoseresidue, mannose residue, N-acetylglucosamine residue,N-acetylgalactosamine residue, xylose residue, lactose residue,N-acetyllactosamine residue, chitobiose residue, etc. Among those,N-acetylglucosamine residue is preferable.

There is no limitation to the bivalent linking group of R⁷ with a lengthequivalent to 2 to 20 methylene groups, and examples thereof includeC₂₋₂₀ (preferably C₄-C₁₈) alkylene groups, etc. Examples of C₂₋₂₀alkylene groups include ethylene, butylene, hexylene, dodecylene,octadecylene, etc.

There is no limitation to the bivalent linking group of R⁸ with a lengthequivalent to 5 to 19 methylene groups, and examples thereof includeC₅₋₁₉ (preferably C₅-C₁₅) alkylene groups, etc. Examples of C₅₋₁₉alkylene groups include pentylene group, heptylene group, undecylengroup, heptadecylene group, etc.

There is no limitation to a peptide residue bound to the monosaccharideor oligosaccharide residue, which is bound to a side chain of awater-soluble polymer through a linker having a selectively cleavablebond; however, a peptide residue composed of 2 to 30 amino acid residuesbound to a monosaccharide or an oligosaccharide residue is preferable,and a peptide residue composed of 4 to 20 amino acid residues bound to amonosaccharide or an oligosaccharide residue is particularly preferable.There is no limitation to the constituent amino acid residues as long asthey have an amino group and a carboxyl group in the molecule. Examplesthereof include Gly (glycine), Ala (alanine), Val (valine), Leu(leucine), Ile (isoleucine), Tyr (tyrosine), Phe (phenylalanine), Trp(tryptophan), Glu (glutamic acid), Asp (aspartic acid), Lys (lysine),Arg (arginine), His (hystidine), Cys (cystein), Met (methionine), Ser(serine), Thr (threonine), Asn (asparagine), Gln (glutamine), Pro(proline) and like α-amino acid residues, and β-Ala and like β-aminoacid residues, etc. Such amino acid residues may be D-amino acid orL-amino acid residues; however, L-amino acid residues are preferable.

There is no limitation to the monosaccharide residue or oligosaccharideresidue bound, and examples thereof include galactose residue, mannoseresidue, N-acetylglucosamine residue, N-acetylgalactosamine residue,glucose residue, xylose residue, sialic acid residue,N-acetyllactosamine residue, lactose residue, chitobiose residue,α-2,3-sialyllactosamine residue,3-β-galactosyl-(6-β-N-acetylglucosaminyl)-N-acetylgalactosamine residue,etc. Such monosaccharide residues or oligosaccharide residues may bebound by either α-linkages or β-linkages. Here, sialic acid is a generalterm for acyl derivatives of neuraminic acid, and includesN-acetylneuramic acid, N-glycolylneuraminic acid,9-O-acetyl-N-acetylneuramic acid, etc.

Examples of linkers preferably used in the present invention includegroups represented by General Formula (IV)—R⁹-R¹⁰—  (IV)wherein R⁹ is a bivalent linking group with a length equivalent to 1 to20 methylene groups, and R¹⁰ is an amino acid residue or a peptideresidue having a site cleavable by a specific protease.

Examples of amino acid residues and peptide residues to which amonosaccharide residue or an oligosaccharide is bound include residuescontaining at least one amino acid selected from the group consisting ofSer, Thr, Glu, Gln, Asp, Asn, Lys, Cys, and Tyr; and a side chainfunctional group of such an amino acid can be bound to a sugar residuedirectly or via a bivalent linking group.

There is no limitation to the bivalent linking group of R⁹ with a lengthequivalent to 1 to 20 methylene groups, and examples thereof includegroups represented by General Formula (V)-A-(CH₂)_(n)—CO—  (V)wherein A is O, CH₂, C═O or NH, the group is linked to a side chain of awater-soluble polymer through A, and n is an integer from 1 to 18.Specific examples thereof are as follows:

-   -   —CH₂—CH₂—CO— —CH₂— (CH₂)₇—CO—    -   —CH₂—(CH₂)₁₁—CO— —O—(CH₂)₆—CO—    -   —O— (CH₂)₁₂—CO— —NH—(CH₂)₆—CO—    -   —NH—(CH₂)₁₂—CO— —CO—CH₂—CO—    -   —CO—(CH₂)₅—CO— —CO—(CH₂)₁₁—CO—    -   —CO— (CH₂)₁₇—CO—

There is no limitation to the amino acid or peptide residue of R¹⁰having a cleavable site by an appropriate protease as long the aminoacid or peptide residue comprises a site that can be cleaved by aprotease that does not contain a cleavage site in an amino acid or apeptide residue to which a monosaccharide or an oligosaccharide residueis bound. For example, when the appropriate protease is α-chymotrypsin,R¹⁰ may be phenylalanine, tryptophan, tyrosine or a like aromatic aminoacid residue, when the appropriate protease is proline-specificprotease, R¹⁰ may be a proline residue, when the appropriate protease islysine-specific protease, R¹⁰ may be a lysine residue, when theappropriate protease is glutamic acid-specific protease, R¹⁰ may be aglutamic acid residue, when the appropriate protease is trypsin, R¹⁰ maybe arginine, lysine or a like basic amino acid residue, when theappropriate protease is Factor Xa, R¹⁰ may be an Ile (isoleucine)-Glu(glutamic acid) or Asp (aspartic acid)-Gly (glycine)-Arg (arginine)residue, and when the appropriate protease is enterokinase, R¹⁰ may bean Asp(aspartic acid)-Asp(aspartic acid)-Asp(aspartic acid)-Asp(asparticacid)-Lys(lysine) residue, etc. R¹⁰ maybe suitably selected depending onthe type of the amino acid or peptide residue to which a monosaccharideor an oligosaccharide residue is bound.

There is no limitation to the amino acid residue having a monosaccharideor an oligosaccharide residue bound to a side chain functional groupthrough a bivalent linking group via a glycosidic bound, as long as theamino residue comprises a side chain functional group that can bond amonosaccharide or an oligosaccharide residue through a bivalent linkinggroup via a glycosidic linkage; however, Ser, Thr, Lys, Asp, Glu, Tyr,Cys, Asn and Gln residues are preferable.

There is no limitation to the bivalent linking group as long as it canlink an amino acid residue to a monosaccharide residue, but a bivalentlinking group with a length equivalent to 1 to 20 methylene groups ispreferable, and especially preferable is a group represented by GeneralFormula (VI)—B—(CH₂)_(n)—O—  (VI)wherein B is O, NH, or C═O, the group is linked to a side chainfunctional group of an amino acid residue through B, and n is an integerfrom 1 to 18. Specific examples thereof are as follows:

-   -   —O—(CH₂)₆—O— —O—(CH₂) 12-O—    -   —NH—(CH₂) 6-O— —NH—(CH₂)₁₂—O—    -   —CO—(CH₂)₃—O— —CO—(CH₂)₅—O—    -   —CO—(CH₂)₁₁—O— —CO— (CH₂)₁₇—O—.

In one preferable embodiment, the sugar chain-having polymer of thepresent invention comprises a side chain functional group and is acopolymer of three or more types of monomers obtained bycopolymerization (i) a polymerizable vinyl monomer to which amonosaccharide or an oligosaccharide residue, or an amino acid orpeptide residue bonded to a monosaccharide or an oligosaccharide residueis bound to a side chain functional group of the polymer through alinker comprising a selectively cleavable bond, (ii) (meth)acrylic acid,and (iii) at least one of other type of vinyl monomer. The proportion of(meth)acrylic acid in total vinyl-based copolymer is 20 to 80 mol %, andpreferably 40 to 60 mol %. Furthermore, the proportion of thepolymerizable vinyl monomers that have a side chain functional group towhich a monosaccharide or an oligosaccharide residue, or an amino acidor peptide residue bound to a monosaccharide or an oligosaccharideresidue is linked through a linker comprising a selectively cleavablebond in the total monomer is not limited, but is preferably 0.1 to 50mol % and more preferably 1 to 25 mol %.

Examples of polymerizable vinyl monomers having a side chain functionalgroup to which a monosaccharide residue, an oligosaccharide residue, oran amino acid or a peptide residue bound with a monosaccharide or anoligosaccharide residue is linked through a linker comprising aselectively cleavable bond include acrylamide derivatives represented byGeneral Formula (VII) wherein R¹¹ is a monosaccharide or anoligosaccharide residue, R¹² is a bivalent linking group with a lengthequivalent to 4 to 20 methylene groups; acrylamide derivativesrepresented by General Formula (IX) wherein R¹⁷ is a linear or branchedC₆₋₂₀ alkyl or alkenyl group, and n is an integer from 5 to 19;acrylamide derivatives represented by General Formula (X) wherein R¹⁸ isa C₂₋₂₀ alkylene group, and R¹⁹ is a C₅₋₁₉ alkylene group; andacrylamide derivatives represented by General Formula (XI) wherein R²⁰is a C₁₋₁₈ alkylene group, R²¹ is an amino acid or a peptide residuehaving a cleavable site by a appropriate protease, and R²² is a Ser,Thr, Glu, Gln, Asp, Asn, Lys, Cys, or Tyr residue to which amonosaccharide or an oligosaccharide residue is bound directly orthrough a bivalent linking group via a glycosidic bond or is a peptideresidue containing such an amino acid residue.

Note that instead of the acrylamide derivatives represented by GeneralFormulae (VII), (IX), (X), and (XI), it is also possible to use thecorresponding methacrylamide derivatives. The correspondingmethacrylamide derivative can be produced by using the same materialsand employing the same methods for producing acrylamide derivatives asdescribed below except for using methacrylamide instead of acrylamide.

The copolymerization can be conducted using techniques such as radicalpolymerization, cationic polymerization, anionic polymerization, etc.Among such techniques, radical polymerization using ammoniumperoxodisulfate, etc., as a catalyst is preferably employed.

Examples of vinyl monomers include acrylamide derivatives,methacrylamide derivatives, methacrylic acid, acrylic esters,methacrylic esters, styrene derivatives, fatty acid vinyl esters, etc.

Examples of acrylamide derivatives include acrylamide, andN-methylacrylamide, N-ethylacrylamide, N-isopropylacrylamide and likeN-alkylacrylamides.

Examples of methacrylamide derivatives include methacrylamide, andN-methylmethacrylamide, N-isopropylmethacrylamide and the likeN-alkylmethacrylamides, etc.

Examples of acrylic esters include methyl acrylate, ethyl acrylate,hydroxyethyl acrylate, and dimethylaminoethyl acrylate.

Examples of methacrylic esters include methyl methacrylate, ethylmethacrylate, hydroxyethyl methacrylate, dimethylaminoethylmethacrylate, etc.

Examples of styrene derivatives include styrene, p-hydroxystyrene, etc.

Examples of fatty acid vinyl esters include vinyl acetate, vinylbutyrate, etc.

The molecular weight of such a copolymer is usually about 10000 to about10000000, preferably 20000 to 5000000, and more preferably 50000 to2000000.

The acrylamide derivatives represented by General Formula (VII), (IX),(X), and (XI) can be synthesized by various techniques commonly employedin synthetic organic chemistry.

The acrylamide derivative represented by General Formula (VII) can beobtained by condensing, in the presence of a suitable catalyst,p-nitrobenzyl alcohol with a sugar oxazoline derivative represented byGeneral Formula (XII) (wherein R²³, R²⁴, and R²⁵ are each independentlyan acyl-type protecting group, ether-type protecting group or amonosaccharide or an oligosaccharide residue having a hydroxyl groupprotected with an acyl-type protecting group and/or an ether-typeprotecting group), a halogenated sugar represented by General Formula(XIII) (wherein X¹ is F, Cl, Br or OC(NH)CCl₃, R²⁶ is a grouprepresented by NHCOCH₃ or OR³⁰, and R²⁷, R²⁸, R²⁹ and R³⁰ are eachindependently an acyl-type protecting group, an ether-type protectinggroup, or a monosaccharide or an oligosaccharide residue having ahydroxyl group protected with an acyl-type protecting group and/orether-type protecting group), or a trichloroacetimidate derivativerepresented by General Formula (XIII); converting the nitro group intoan amino group by reduction; and condensing the resultant with anacrylamide derivative represented by General Formula (XV) (wherein n isan integer from 4 to 20) obtained by condensing an ω-amino fatty acidrepresented by General Formula (XIV) (wherein n an integer from 4 to 20)with a acryloyl chloride in the presence of a suitable condensing agent.

The acrylamide derivative represented by General Formula (IX) can beobtained by, for example, condensing an activated lactose derivativerepresented by General Formula (XVI) (wherein R³¹, R³², R³³, R³⁴, R³⁵,R³⁶ and R³⁷ are each independently an acyl-type protecting group or anether-type protecting group, and X² is F, Cl, Br, or OC(NH)CCl₃) and aserine derivative represented by General Formula (XVII) (wherein R³⁸ isa C₆₋₂₀ alkyl group or an alkenyl group, and R³⁹ is a protecting group)in the presence of suitable catalyst; removing the protecting group ofthe amino group in the serine residue; condensing with an acrylamidederivative represented by General Formula (XVIII) (wherein Y¹ is OH, Cl,or Br, and n is an integer from 5 to 9); and removing the protectinggroups in lactose moiety.

The acrylamide derivative represented by General Formula (X) can beobtained by condensing, in the presence of a suitable catalyst, a sugaroxazoline derivative represented by General Formula (XIX) (wherein R⁴⁰,R⁴¹, and R⁴² each independently acyl-type protecting group or ether-typeprotecting group) with a phenylalanine derivative represented by GeneralFormula (XX) (wherein R⁴³ is a C₂₋₂₀ alkylene group and R⁴⁴ is aprotecting group), removing the protecting group, condensing with anacrylamide derivative represented by General Formula (XXI) (wherein R⁴⁵is a C₅₋₁₉ alkylene group, and Y² is OH, Cl, or Br), and removing theprotecting groups of the sugar portion.

The acrylamide derivative represented by General Formula (XI) can besynthesized using automated peptide synthesizer. Here below is explainedwhen the protease used is α-chymotrypsin, R²¹ is an aromatic amino acidresidue, R²² is an arbitrary peptide residue which does not contain anaromatic amino acid and which contains a serine residue, a threonineresidue, a glutamine residue or an asparagines residue to which amonosaccharide residue is bound via a glycosidic bond formed between anOH group or an acid amide group and a monosaccharide residue, or apeptide residue contains an amino acid residue to which a monosaccharideresidue is linked to a side chain functional group via a glycosidic bondthrough a bivalent linking group. First, peptide chain is elongated on asuitable solid-phase carrier to synthesize an arbitrary peptide thatdoes not contain aromatic amino acid residue, but contains a serineresidue, a threonine residue, a glutamine residue, or an asparagineresidue to which a monosaccharide residue is bound via a glycosidic bondformed between an OH position or an acid amide group and amonosaccharide rsidue, or a peptide containing an amino acid residue towhich an arbitrary monosaccharide residue is linked to a side chainfunctional group via a glycosidic bond through a bivalent linking group.Second, the peptide chain is elongated using an aromatic amino acidderivative in which an amino group is acylated by a group represented byGeneral Formula (XXII) (wherein R⁴⁶ is a C₁₋₁₈ alkylene group), theelongated peptide is released from the solid-phase carrier, and aprotecting groups in the peptide chain and monosaccharide residue areremoved.

The peptide residue containing a serine residue, a threonine residue, aglutamine residue or an asparagine residue to which a monosaccharideresidue is bound via a glycosidic bond formed between an OH position oran acid amide group and a monosaccharide residue, or the peptide residuecontaining an amino acid residue to which a monosaccharide residue islinked to a side chain functional group by a glycosidic bond through abivalent linking group, can be introduced by using, instead of astandard N-protected amino acid, the corresponding N-protected aminoacid to which a monosaccharide residue having hydroxyl groups protectedwith suitable protecting groups is bound to a side chain functionalgroup via a glycosidic bond formed between an OH position or an acidamide group and a monosaccharide residue, or a bivalent linking group.An aromatic amino acid in which an amino group is acylated by a grouprepresented by General Formula (XXII) can be also introduced byemploying the same methods as for introducing typical N-protected aminoacids.

Serine, threonine, glutamine and asparagine residues to which amonosaccharide residue having hydroxyl groups protected with suitableprotecting groups is bound via a glycosidic bond formed between an OHposition or acid amide and a monosaccharide residue, and N-protectedamino acids to which a monosaccharide residue having hydroxyl groupsprotected with suitable protecting groups bonded to a side chainfunctional group through a bivalent linking group via a glycosidiclinkage can be obtained by employing commonly used synthetic organicchemistry techniques. Furthermore, some N-protected amino acids to whicha monosaccharide residue is bound, for example,Fmoc-Asn(GlcNAc(Ac)3-β-D)-OH, Fmoc-Ser(GalNAc(Ac)3-α-D)-OH,Fmoc-Thr(GalNAc(Ac)3-α-D)-OH, etc., are already commercially availableand therefore it is possible to use these.

A polymerizable aromatic amino acid derivative in which an amino groupis acylated by a group represented by General Formula (XXII) can besynthesized by employing commonly used synthetic organic chemistrytechniques. For example, when the aromatic amino acid residue is aphenylalanine residue, the polymerizable phenylalanine derivative can beprepared by condensing a phenylalanine ethyl ester with anω-acryloylamino fatty acid, and hydrolyzing the ethyl ester.Condensation of phenylalanine ethyl ester with an ω-acryloylamino fattyacid is not limited as long as phenylalanine ethyl ester and theω-acryloylamino fatty acid can be condensed, and they can be condensedby contacting in the presence of a condensation agent typically used forpeptide-bond formation, such as dicyclohexylcarbodiimide,carbodiimidazole, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline,diphenylphosphoryl azide, etc.

In the first step of producing the glycoconjugate of the presentinvention, a sugar residue is transferred from a sugar nucleotide to asugar chain-having polymer by contacting the sugar chain-havingwater-soluble polymer compound with a glycosyltransferase in thepresence of a sugar nucleotide.

Transfer of a sugar from a sugar nucleotide to a sugar chain-havingpolymer is usually performed by contacting them with aglycosyltransferase in a neutral buffer solution containing a sugarchain-having polymer and sugar nucleotides usually at 10 to 60° C. andpreferably at 20 to 40° C., and usually for 1 to 120 hours andpreferably for 2 to 72 hours. The amount of sugar nucleotides usedrelative to one equivalent of sugar chain-having polymer is preferablyfrom about one equivalent to excess.

Metal salts may be added to the reaction solution if needed. Examples ofusable metal ions include magnesium, manganese, cobalt, nickel, copper,zinc, etc., and these are usually added in a form of chlorides, etc.

The glycosyltransferase used in the present invention is not limited aslong as a sugar nucleotide can be used as a sugar donor. Leloir pathwayglycosyltransferases are examples of such enzymes. Specific examplesthereof include galactosyltransferases, N-acetylglucosaminyltransferase,N-acetylgalactosaminyltransferase, fucosyltransferase,sialyltransferase, mannosyltransferase, xylosyltransferase,glucuronyltransferase, etc. Such enzymes may be free or immobilized,although immobilized enzymes are preferable.

The sugar nucleotide used in the present invention is not limited aslong as the above-mentioned enzyme can be used. Examples of such sugarnucleotides include uridine-5′-diphospho-galactose,uridine-5′-diphospho-N-acetylglucosamine,uridine-5′-diphospho-N-acetylgalactosamine,uridine-5′-diphospho-glucuronic-acid, uridine-5′-diphospho-xylose,guanosine-5′-diphospho-fucose, guanosine-5′-diphospho-mannose,cytidine-5′-monophoshpho-N-acetylneuramic acid, sodium salts thereof,etc.

In the second step of producing the glycoconjugate of the presentinvention, the sugar chain is elongated by transferring a plurality ofsugar residues by repeating step two or more times according torequirements.

In the third step of producing the glycoconjugate of the presentinvention, by-product nucleotides or unreacted sugar nucleotides areremoved if necessary. Methods for removing by-product nucleotides orunreacted sugar nucleotides are not limited as long as the sugarchain-having polymer can be separated from nucleotides and sugarnucleotides. Examples of such methods include gel filtrationchromatography, ion-exchange chromatography, dialysis, ultrafiltration,etc.

In the fourth step of producing the glycoconjugate of the presentinvention, after repeating steps 1 to 3 several times, a glycoconjugateis released from the sugar chain-having polymer whose sugar chain hasbeen elongated by transferred sugar residues. The method for release theglycoconjugate whose sugar chain has been elongated from the sugarchain-having polymer of the present invention is not limited as long asit can be released without decomposing the glycoconjugate whose sugarchain has been elongated. Examples of such methods include releasingunder weak acid or weak alkali, by hydrogenolysis, by photoreaction, andby enzymatic reaction. Preferable example include hydrogenolysis,oxyzation using 2,3-dichloro-5,6-dicyanobenzoquinone, hydrolysis using aprotease, transfer reaction using ceramide glycanase, etc.

BEST MODE FOR CARRYING OUT THE INVENTION

The following Examples are intended to illustrate the present inventionin further detail, and not to limit the scope of the invention.

REFERENCE EXAMPLE 1 Synthesis of2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline

To a solution of2-acetamide-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranoside (6.0 g) in1,2-dichloroethane (40 ml) was added trimethylsilyltrifluoromethanesulfonic acid (3.2 ml). The mixture was stirred at 50°C. for 7 hours. After the reaction, the reaction mixture was cooled toroom temperature, and triethylamine (10.8 ml) was added. Afterconcentrating the reaction mixture under reduced pressure, the targetcompound was separated by silica gel column chromatography (eluant:toluene/ethyl acetate/triethylamine=100/200/1). The eluate wasevaporated to give the target compound (5.0 g).

REFERENCE EXAMPLE 2 Synthesis ofp-nitrobenzyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside

To a solution of2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline(2.8 g) obtained in Reference Example 1 in dichloroethane (40 ml) wereadded p-nitrobenzyl alcohol (10.4 g) and D-camphor-10-sulfonic acid (0.2g). The mixture was stirred at 80° C. for 2 hours. After the reaction,the reaction mixture was cooled to room temperature, and triethylamine(4.0 ml) was added. After concentrating the reaction mixture underreduced pressure, the target compound was separated by silica gel columnchromatography (eluant: chloroform/ethyl acetate/methanol=200/40/5). Theeluate was evaporated to give the target compound (3.7 g).

REFERENCE EXAMPLE 3 Synthesis of 6-acryloylaminocaproic Acid

To a solution of 6-Aminocaproic acid (10.0 g) in a 1.27 M aqueous sodiumhydroxide solution (60 ml) was added a solution of acryloyl chloride(7.8 ml) in tetrahydrofuran (20 ml) dropwisely while chilling in icewater. During the addition, the reaction mixture was adjusted to be pH 8to 9 by addition of 4 N aqueous sodium hydroxide solution. After theaddition, the resulting mixture was stirred for 2 hours while beinggradually cooled to room temperature. Subsequently, the reaction mixturewas adjusted to be pH 3 by addition of 1 N hydrochloric acid andextracted with ethyl acetate. The organic layer was washed withdistilled water and dried over anhydrous magnesium sulfate. Themagnesium sulfate was filtered off, and the filtrate was concentratedunder reduced pressure. The residue was dissolved in a small amount ofethyl acetate and recrystallized from hexane to give the target compound(9.6 g).

REFERENCE EXAMPLE 4 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside

To a solution ofp-nitrobenzyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside(1.6 g) obtained in Reference Example 2 in methanol (50 ml) were addedammonium formate (2.1 g) and 10% palladium-carbon (170 mg). Afterstirring at room temperature for 5 minutes, the catalyst was filteredoff, and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in chloroform. The mixture was washed withdistilled water and dried over anhydrous sodium sulfate. After thedrying, the sodium sulfate was filtered off, and the filtrate wasconcentrated under reduced pressure. To a solution of the residue inmixed solvent (44 ml) of dichloroethane:N,N-dimethylformamide=10:1 wasadded 0.6 g of the 6-acryloylaminocaproic acid obtained in ReferenceExample 3. Triethylamine (0.46 ml) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (65 mg) werethen added with stirring at 0° C. The reaction mixture was cooled toroom temperature with stirring, and stirred for 22 hours. Chloroform (60ml) was added to the reaction mixture, and the resulting mixture waswashed with 1 N aqueous sodium hydroxide solution, saturated aqueoussodium hydrogencarbonate solution and saturated saline solution in thatorder, and dried over anhydrous sodium sulfate. After filtering off thesodium sulfate and concentrating the filtrate under reduced pressure,the target compound was separated by silica gel column chromatography(eluant: chloroform/ethanol=30/1). The eluate was evaporated to give thetarget compound (1.1 g).

EXAMPLE 1 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside

To a solution ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside(660 mg) obtained in Reference Example 4 in methanol (70 ml) was addedsodium methoxide (50 mg). The reaction mixture was stirred at roomtemperature for 15 hours. After the reaction, an H⁺ cation exchangeresin, Dowex 50WX-8 (Dow Chemical Co.), was added until pH 7. The ionexchange resin was filtered off, and the filtrate was evaporated toafford the target compound (520 mg).

EXAMPLE 2 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicAcid/Acrylamide Copolymer (Copolymerization Ratio=1:2:7, SugarChain-Having Polymer A)

A solution of thep-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside(61.7 mg) obtained in Example 1, acrylic acid (18.0 mg) and acrylamide(62.2 mg) in mixed solvent (1 ml) of dimethyl sulfoxide:distilledwater=3:1 was deaerated well by a water aspirator, to which were addedN,N,N′,N′-tetramethylethylenediamine (hereinafter referred to as TEMED)(11.6 μl) and ammonium peroxodisulfate (8.6 mg). The mixture was stirredat room temperature for 24 hours for copolymerization. After thereaction, the mixture was diluted with distilled water (2 ml), directlysubjected to gel filtration chromatography on Sephadex G-50 (AmershamPharmacia) column and eluted with 50 mM ammonium acetate. The voidfractions were collected and lyophilized to afford the target compound(137 mg).

EXAMPLE 3 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicAcid/Acrylamide Copolymer (Copolymerization Ratio=1:4:5, SugarChain-Having Polymer B)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid and acrylamide were used in amounts of 36.0 mg and44.4 mg, respectively, to afford 138 mg of the target compound.

EXAMPLE 4 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicAcid/Acrylamide Copolymer (Copolymerization Ratio=1:6:3, SugarChain-Having Polymer C)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid and acrylamide were used in amounts of 54.0 mg and26.7 mg, respectively, to afford 139 mg of the target compound.

EXAMPLE 5 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicAcid/Acrylamide Copolymer (Copolymerization Ratio=1:8:1, SugarChain-Having Polymer D)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid and acrylamide were used in amounts of 72.1 mg and 8.9mg, respectively, to afford 139 mg of the target compound.

REFERENCE EXAMPLE 5 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylamideCopolymer (Copolymerization Ratio=1:9, Sugar Chain-Having Polymer E)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid was not used and that acrylamide was used in an amountof 80.0 mg, to afford 138 mg of the target compound.

REFERENCE EXAMPLE 6 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicAcid Copolymer (Copolymerization Ratio=1:9, Sugar Chain-Having PolymerF)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylamide was not used and that acrylic acid was used in an amountof 81.1 mg, to afford 139 mg of the target compound.

EXAMPLE 6 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicacid/N-isopropylacrylamide Copolymer (Copolymerization Ratio=1:2:7,Sugar Chain-Having Polymer G)

Copolymerization was performed in the same manner as in Example 2 exceptfor using 99.0 mg of N-isopropylacrylamide in place of acrylamide, toafford 174 mg of the target compound.

EXAMPLE 7 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicacid/N-isopropylacrylamide Copolymer (Copolymerization Ratio=1:4:5,Sugar Chain-Having Polymer H)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid was used in an amount of 36.0 mg and that 70.7 mg ofN-isopropylacrylamide was used in place of acrylamide, to afford 164 mgof the target compound.

EXAMPLE 8 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicacid/N-isopropylacrylamide Copolymer (Copolymerization Ratio=1:6:3,Sugar Chain-Having Polymer I)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid was used in an amount of 54.0 mg and that 42.4 mg ofN-isopropylacrylamide was used in place of acrylamide, to afford 154 mgof the target compound.

EXAMPLE 9 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylicacid/N-isopropylacrylamide Copolymer (Copolymerization Ratio=1:8:1,Sugar Chain-Having Polymer J)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid was used in an amount of 72.1 mg and that 14.1 mg ofN-isopropylacrylamide was used in place of acrylamide, to afford 144 mgof the target compound.

REFERENCE EXAMPLE 7 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/N-isopropylacrylamideCopolymer (Copolymerization Ratio=1:9, Sugar Chain-Having Polymer K)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid was not used and that N-isopropylacrylamide was usedin an amount of 127.3 mg, to afford 183 mg of the target compound.

REFERENCE EXAMPLE 8 Synthesis ofp-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranoside/acrylamide/N-isopropylacrylamideCopolymer (Copolymerization Ratio=1:5:4, Sugar Chain-Having Polymer L)

Copolymerization was performed in the same manner as in Example 2 exceptthat acrylic acid was not used and that acrylamide andN-isopropylacrylamide were used in amounts of 44.4 mg and 56.6 mg,respectively, to afford 158 mg of the target compound.

REFERENCE EXAMPLE 9 Preparation of Immobilizedβ1,4-galactosyltransferase

0.5 g of CNBr-activated Sepharose 4B (Pharmacia) was washed with threeportions of 1 mM hydrochloric acid (total 100 ml). Five milliliters of0.1 M boric acid buffer solution (pH 8.0) containing 10 U ofβ1,4-galactosyltransferase (Toyobo Co., Ltd.), 30 mg of bovine serumalbumin (hereinafter BSA), 1 mM uridine-5′-diphosphogalactose(hereinafter UDP-Gal), 5 mM N-acetylglucosamine, 25 mM manganesechloride and 0.5 M NaCl was added, and incubated with gently shaking at4° C. overnight. The immobilized β1,4-galactosyltransferase wascollected by filtration with a glass filter, and washed with 5 ml of thesame buffer solution as above except for containing noβ1,4-galactosyltransferase. Five milliliters of 0.1 M Tris-HCl buffersolution (pH 8.0) was added to block remained active groups on thesupport. After washing with 1 M aqueous sodium chloride solution andthen with distilled water, the immobilized β1,4-galactosyltransferasewas immersed in 25 mM cacodylate buffer (pH 7.4) containing 1 mM UDP-Galand 5 mM manganese chloride, and stored at 4° C. The obtainedimmobilized enzyme had an activity of 1.5 U/ml.

EXAMPLE 10 Galactose Transfer to Sugar Chain-Having Polymers byImmobilized β1,4-galactosyltransferase

To 2.0 ml of 50 mM HEPES buffer solution (pH 7.5) containing 10 mMuridine-5′-diphosphogalactose, 10 mM manganese chloride and 0.26 mg/mlof α-lactoalbumin were added 1 ml of the immobilizedβ1,4-galactosyltransferase obtained in Reference Example 9 and 20 mg ofone of sugar chain-having polymers A to L obtained in Examples 2 to 9and Reference Examples 5 to 8, and the mixture was incubated withshaking at 37° C. for 24 hours. The reaction mixture was centrifuged,and the supernatant was subjected to Sephadex G-25 column chromatography(eluant: distilled water). The void fractions were then lyophilized toafford 19 mg of a product. To a solution of the product (1 mg) in 1 mlof mixed solvent of distilled water:ethanol=3:1 was added 1 mg of 10%palladium-carbon, and the nmixture was stirred under hydrogen atmosphereat room temperature for 24 hours. After filtering off the catalyst, thefiltrate was further filtered with an ultrafiltration unit, Ultra FreeMC (molecular weight cut-off: ca. 10,000, Millipore Corp.), to therebycollect the released oligosaccharide as a permeated fraction. Thepermeated fraction was lyophilized, and the rsidue was pyridylaminatedby standard method and subjected to HPLC to analyze the proportions ofN-acetyllactosamine and N-acetylglucosamine and thereby determine thesugar transfer yield. These results showed that the galactose transferto each polymer proceeded quantitatively.

REFERENCE EXAMPLE 10 Preparation of Immobilized α2,3-sialyltransferase

0.5 g of NHS-activated Sepharose (Amersham Pharmacia) was washed withthree portions of 1 mM hydrochloric acid (total 100 ml). Fivemilliliters of 50 mM HEPES buffer (pH 7.5) containing 1 U of pig liverα2,3-sialyltransferase, 30 mg of BSA and 1 mM cytidine-5′-diphosphatewas added and incubated with gently shaking at 4° C. overnight. Theimmobilized α2,3-sialyltransferase was collected by filtration through aglass filter, and washed with 5 ml of the same buffer solution as aboveexcept for containing no α2,3-sialyltransferase. Five milliliters of 0.1M Tris-HCl buffer (pH 8.0) was added to block remaining active groups onthe support. After further washing, the α2,3-sialyltransferase wasimmersed in 25 mM cacodylate buffer (pH 7.4) containing 1 mMcytidine-5′-monophospho-N-acetylneuraminic acid (hereinafter CMP-NeuAc),and stored at 4° C. The obtained immobilized enzyme had an activity of110 mU/ml.

EXAMPLE 11 Sialic Acid Transfer to Sugar Chain-Having Polymers byImmobilized α2,3-sialyltransferase

To 0.5 ml of 50 mM HEPES buffer (pH 7.0) containing 0.05 ml of theimmobilized α2,3-sialyltransferase obtained in Reference Example 10, 50mM CMP-NeuAc, 10 mM manganese chloride and 0.1% of Triton CF-54 wasadded one of galactosylated sugar chain-having polymers A to L obtainedin Example 10 in an amount of 6.5 mg (sugar chain-having polymer A toF), 8.0 mg, 7.5 mg, 7.1 mg, 6.7 mg, 8.4 mg or 7.3 mg, respectively,(corresponding to 5 μmol of N-acetyllactosamine residues), and themixture was incubated with shaking at 30° C. for 24 hours. After theincubation, the reaction mixture was centrifuged, and a product wasseparated from the supernatant in the same manner as in Example 10.Thus, 6.0 mg (sugar chain-having polymers A to F), 7.5 mg, 7.0 mg, 6.5mg, 6.2 mg, 7.8 mg or 6.8 mg of the product was obtained, respectively.One milligram of the product was weighed out, and the sugar transferyield was determined in the same manner as in Example 10. TABLE 1Results of sugar transfer reactions by immobilizedα2,3-sialyltransferase Sugar chain- Vinyl monomer contents havingpolymer GM AA AAm NIPAM Yield A 10 20 70 0 69% B 10 40 50 0 74% C 10 6030 0 70% D 10 80 10 0 64% E 10 0 90 0 15% F 10 90 0 0 52% G 10 20 0 7073% H 10 40 0 50 76% I 10 60 0 30 86% J 10 80 0 10 71% K 10 0 0 90 40% L10 0 50 40 29%GM:p-N-(6-acryloylaminohexanoyl)amino-benzyl-2-acetamide-2-deoxy-D-glucopyranosideAA: Acrylic acidAam: AcrylamideNIPAM: N-isopropylacrylamide

REFERENCE EXAMPLE 11 Synthesis ofp-nitrobenzyl-4-(2′,3′,4,6′-tetra-O-acetyl-D-galactopyranosyl)-2,3,6-tri-O-acetyl-D-glucopyranoside

To a solution of1-bromo-4-(2′,3′,4′,6′-tetra-O-acetyl-D-galactopyranosyl)-2,3,6-tri-O-acetyl-D-glucopyranoside(5.0 g) in dichloroethane (50 ml) were added p-nitrobenzyl alcohol (23.5g) and molecular sieves 4 Å (5.0 g). Silver triflate (2.9 g) was addedunder a stream of nitrogen with stirring at 0° C. After addition, thereaction mixture was gradually allowed to room temperature, and stirredfor 12 hours. The reaction mixture was diluted with chloroform andfiltered with celite. The filtrate was washed with saturated salinesolution, and the organic layer was dried over anhydrous magnesiumsulfate. The magnesium sulfate was filtered off, and the filtrate wasconcentrated under reduced pressure and subjected to silica gelchromatography (eluant: chloroform/methanol=50/1). The eluate wasevaporated to give the target compound (5.6 g).

REFERENCE EXAMPLE 12 Synthesis ofp-N-(6-acryloylaminohexanoyl)-benzyl-4-(2′,3′,4′,6′-tetra-O-acetyl-D-galactopyranosyl)-2,3,6-tri-O-acetyl-D-glucopyranoside

To a solution of thep-nitrobenzyl-4-(2′,3′,4′,6′-tetra-O-acetyl-D-galactopyranosyl)-2,3,6-tri-O-acetyl-D-glucopyranoside(3.0 g) obtained in Reference Example 11 in methanol (50 ml) were addedammonium formate (1.8 g) and 10% palladium-carbon (200 mg). Afterstirring at room temperature for 5 minutes, the catalyst was filteredoff, and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in chloroform. The mixture was washed withdistilled water and dried over anhydrous magnesium sulfate. After thedrying, the magnesium sulfate was filtered off, and the filtrate wasconcentrated under reduced pressure. To a solution of the residue inmixed solvent of dichloroethane:N,N,-dimethylformamide=10:1 (40 ml) wasadded 6-acryloylaminocaproic acid (0.85 g) obtained in Reference Example3. Subsequently, triethylamine (634 μl) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (870 mg)were added with stirring and chilling in ice water, and the reactionmixture was allowed to room temperature with stirring. After stirringfor 22 hours the reaction mixture was diluted with chloroform, andwashed sequentially with 1 N sulfuric acid, saturated aqueous sodiumbicarbonate solution and saturated saline solution. The organic layerwas dried over anhydrous magnesium sulfate, the magnesium sulfate wasfiltered off, and the filtrate was concentrated under reduced pressureand subjected to silica gel chromatography (eluant:chloroform/ethanol=20/1). The eluate was evaporated to give the targetcompound (1.1 g).

EXAMPLE 12 Synthesis ofp-N-(6-acryloylaminohexanoyl)-benzyl-4-D-galactopyranosyl-D-glucopyranoside

A solution of thep-N-(6-acryloylaminohexanoyl)-benzyl-2,3,4,6-tetra-O-acetyl-D-galactopyranosyl-2,3,6-tri-O-acetyl-D-glucopyranoside(1.0 g) obtained in Reference Example 12 in methanol (10 mL) was addedsodium methoxide (24 mg). The reaction mixture was stirred at roomtemperature for 15 hours. After the reaction, the reaction mixture wasneutralized with an ion exchange resin, Dowex 50WX8 (H⁺). The resin wasfiltered off, and the filtrate was evaporated to afford the targetcompound (650 mg).

EXAMPLE 13 Synthesis ofp-N-(6-acryloylaminohexanoyl)-benzyl-D-galactopyranosyl-D-glucopyranoside/acrylicAcid/Acrylamide Copolymer (Copolymerization Ratio=1:4:5, SugarChain-Having Polymer M)

Using 76.8 mg of thep-N-(6-acryloylaminohexanoyl)-benzyl-D-galactopyranosyl-D-glucopyranosideobtained in Example 12, 36.0 mg of acrylic acid and 44.4 mg ofacrylamide, copolymerization was performed by following the procedure ofExample 2 to afford 151 mg of the target compound.

EXAMPLE 14 Sialic Acid Transfer to Sugar Chain-Having Polymer M UsingImmobilized α2,3-sialyltransferase

6.3 mg of sugar chain-having polymer M obtained in Example 13(corresponding to 5 μmol of lactose residues) was incubated in 50 mMHEPES buffer (1.0 ml, pH 7.0) containing 0.3 ml of the immobilizedα2,3-sialyltransferase obtained in Reference Example 10, 50 mMCMP-NeuAc, 10 mM manganese chloride, and 0.1% of Triton CF-54 withshaking at 30° C. for 24 hours. After the incubation, the reactionmixture was centrifuged, and a product was separated from supernatant inthe same manner as in Example 10 to thereby afford 5 mg of the product.One milligram of the product was weighed out, and the yield of thesialic acid transfer reaction was determined in the same manner as inExample 10 and found to be 88%.

REFERENCE EXAMPLE 13 Synthesis ofN-(6-acryloylaminocaproyl)phenylalanine Ethyl Ester

To a solution of phenylalanine ethyl ester hydrochloride (1.15 g) and6-acryloylaminocaproic acid (1.11 g) obtained in Reference Example 3 indimethylformamide (15 ml, hereinafter DMF) was added a solution ofdiphenylphosphoryl azide (1.65 g) in DMF (15 ml) with stirring whilechilling in ice water, and a solution of triethylamine (1.11 g) in DMF(15 ml) was added dropwisely thereto. The reaction mixture was stirredwhile chilling in ice water for 4 hours, and then at room temperaturefor 24 hours. After the reaction, 450 ml of mixed solvent ofbenzene:ethyl acetate=1:1 was added, and the organic layer was washedwith 5% hydrochloric acid, distilled water, saturated saline solution,saturated aqueous sodium hydrogencarbonate solution, distilled water andsaturated saline solution in that order. The organic layer was driedover anhydrous sodium sulfate and then concentrated under reducedpressure, and the residue was recrystallized from benzene to give thetarget compound (1.35 g).

REFERENCE EXAMPLE 14 Synthesis ofN-(6-acryloylaminocaproyl)phenylalanine

0.72 g of the N-(6-acryloylaminocaproyl)phenylalanine ethyl esterobtained in Reference Example 13 was added to 50 ml of methanolcontaining 1 N sodium hydroxide, followed by stirring at roomtemperature for 4 hours. After the reaction, an H⁺ cation exchangeresin, Dowex 50W (Dow Chemical Co.), was added for neutralization. Theion exchange resin was then filtered off, and the filtrate wasevaporated to afford the target compound (0.65 g).

REFERENCE EXAMPLE 154-Pentenyl-3′,4′,6′-tri-O-acetyl-N-acetylglucosamine

To a solution of the2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline(3.3 g) obtained in Reference Example 1 and 4-penten-1-ol (1.7 g) in1,2-dichloroethane (40 ml) was added CSA to be pH 2-3. The reactionmixture was stirred at 70° C. for 30 minutes. The reaction mixture wascooled to room temperature, diluted with chloroform and washed twicewith saturated aqueous sodium hydrogencarbonate solution. The organiclayer was dried over anhydrous magnesium sulfate overnight. Themagnesium sulfate was filtered off with celite, and the filtrate wasconcentrated under reduced pressure and subjected to silica gelchromatography (eluant: chloroform). The eluate was evaporated to givethe target compound (2.5 g).

REFERENCE EXAMPLE 164-O-(3′,4′,6′-tri-O-acetyl-N-acetylglucosaminyl)butyric Acid

To a solution of potassium permanganate (1.95 g) in 17% aqueous aceticacid solution (35 ml) was added dropwisely with stirring under chillingin ice water a solution of4-pentenyl-3′,4′,6′-tri-O-acetyl-N-acetylglucosamine (1.6 g) obtained inReference Example 15 in glacial acetic acid (35 ml). The reactionmixture was stirred for 3 hours. After the reaction, ethyl acetate (300ml) was added to the reaction mixture, and sodium sulfate (3.16 g) and 1M hydrochloric acid (35 ml) were further added, and the resultingmixture was stirred while chilling in ice water. The organic layer wasseparated, washed with saturated saline solution and dried overanhydrous magnesium sulfate. After the drying, the magnesium sulfate wasfiltered off, and the filtrate was evaporated to afford the targetcompound (1.5 g).

REFERENCE EXAMPLE 17 Synthesis ofN-α-(9-fluorenylmethyloxycarbonyl)-N-ε-(4-O— (3′,4′,6′-tri-O-acetyl-N-acetylglucosaminyl)butanoyl)lysine

To a solution of the4-O-(3′,4′,6′-tri-O-acetyl-N-acetylglucosaminyl)butyric acid (0.43 g)obtained in Reference Example 16 in chloroform (20 ml) were added whilechilling in icewater N-hydroxysuccinimide (0.12 g) anddicyclohexylcarbodiimide (0.21 g). The reaction mixture was stirredovernight. After the stirring, the reaction mixture was filtered, andthe filtrate was concentrated under reduced pressure. The residue wasdissolved in dimethoxymethane (10 ml), and a solution ofN-α-(9-fluorenylmethyloxycarbonyl)lysine (0.37 g) in dimethoxymethane(10 ml) was added. The resulting mixture was stirred at room temperaturefor 1 hour. Water (100 ml) was poured. The precipitate was collected andwashed with distilled water, 10% aqueous sodium hydrogencarbonatesolution, 1 N hydrochloric acid and distilled water in that order. Afterevaporation, the residue was recrystallized from ethanol to give thetarget compound (0.56 g).N-α-(9-fluorenylmethyloxycarbonyl)-N-ε-(4-O-(3′, 4′,6′-tri-O-acetyl-N-acetylglucosaminyl)butanoyl)lysine had the followingstructural formula, wherein Fmoc is a 9-fluorenylmethyloxycarbonyl groupand Ac is an acetyl group.

REFERENCE EXAMPLE 18 Synthesis of Acrylamide Derivative A

Using 0.44 g of 2-chlorotrityl resin preloaded with Fmoc-Ser(tBu) (0.23mmol of Ser residues were attached per gram of the resin) as a sugarchain-having polymer, 1.0 mmol of each of the following N-protectedamino acids were sequentially condensed by the Fmoc/DCC/HOBt methodusing a peptide synthesizer Model A433 (ABI) to synthesize the targetacrylamide derivative on a solid phase support: Fmoc-Asp(OtBu)-OH,Fmoc-Gly-OH, Emoc-Arg(Pmc)-OH, Fmoc-Gly-OH, Fmoc-Asn(PAc₃GlcNAc)-OH,Fmoc-Gly-OH, N-(6-acryloylaminocaproyl)phenylalanine obtained inReference Example 14. Deprotection of peptide residues was carried outat room temperature for 1 hour in dichloromethane containing 50%trifluoroacetic acid, 1% 1,2-ethanedithiol, 1% thioanisole and 5%phenol, and release the acrylamide derivative from the solid phasesupport. The resin was filtered off, and after concentration underreduced pressure, the residue was diluted with mixed solvent of ethylacetate and chloroform (1:1). The organic layer was then washed withdistilled water. HPLC (column: YMC-Pack ODS 20 mm×250 mm, eluant:A:B=100:0 (0 minutes)-50:50 (60 minutes), A: 0.1% aqueoustrifluoroacetic acid solution, B: 0.1% trifluoroacetic acid acetonitrilesolution, flow rate: 9.0 ml/min) was performed to purify the acrylamidederivative. The acrylamide derivative fraction was lyophilized, and 30ml of methanol containing 2.2 mg of sodium methoxide was added to theresidue, followed by stirring at room temperature for 2 hours. Theresulting mixture was neutralized by adding an H⁺ cation exchange resin,Dowex 50W (Dow Chemical Co.), and the ion exchange resin was thenfiltered off, and the filtrate was evaporated to afford 96 mg of thetarget compound (acrylamide derivative A). Acrylamide derivative A hadthe following structural formula, wherein Ac is an acetyl group.

REFERENCE EXAMPLE 19 Synthesis of Acrylamide Derivative B

Following the procedure of Reference Example 18, 1.0 mmol of each of thefollowing N-protected amino acids were sequentially condensed by theFmoc/DCC/HOBt method to synthesize the target acrylamide derivative on asolid phase support: Fmoc-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg (Pmc)-OH,Fmoc-Gly-OH, N-α-(9-fluorenylmethyloxycarbonyl)-N-ε-(4-O— (3′,4′,6′-tri-O-acetyl-N-acetylglucosaminyl)butanoyl)lysine obtained inReference Example 17, and N-(6-acryloylaminocaproyl)phenylalanineobtained in Reference Example 14. Following the procedure of ReferenceExample 18, the protective groups were removed from the peptideresidues, and the target compound, an acrylamide derivative, wasreleased from the solid phase support and purified to afford 97 mg ofthe target compound (acrylamide derivative B). Acrylamide derivative Bhad the following structural formula, wherein Ac is an acetyl group.

EXAMPLE 15 Synthesis of Acrylamide Derivative A/Acrylic Acid/AcrylamideCopolymer (Copolymerization Ratio=1:4:6, Sugar Chain-Having Polymer N)

To a solution of acrylamide derivative A (60 mg) obtained in ReferenceExample 18 in 2 ml of dimethylsulfoxide (hereinafter DMSO) was added asolution of acrylic acid (14.4 mg) and acrylamide (21.3 mg) in water (1ml). Subsequently, N,N,N′,N′-tetramethylethylenediamine (7.5 μl) andammonium peroxodisulfate (4.5 mg) were added, and copolymerization wascarried out at 50° C. for 24 hours. The reaction mixture wasconcentrated under reduced pressure. After distilling off DMSO, thersidue was subjected to column chromatography on Sephadex G-25(Pharmacia) (eluant: 10 mM ammonium acetate). The void eluant wascollected and lyophilized to afford 90 mg of the target compound (sugarchain-having polymer N). In the obtained polymer, the acrylamidederivative A residues to which a glycopeptide was bound had thefollowing structural formula, wherein Ac is an acetyl group, and werecontained in a proportion of about 9 mol %.

EXAMPLE 16 Synthesis of Acrylamide Derivative B/Acrylic Acid/AcrylamideCopolymer (Copolymerization Ratio=1:4:6, Sugar Chain-Having Polymer O)

A reaction was carried out in the same manner as in Example 15 exceptfor using 61 mg of acrylamide derivative B obtained in Reference Example19 in place of 60 mg of acrylamide derivative A obtained in ReferenceExample 18, to afford 91 mg of the target compound (sugar chain-havingpolymer O). In the obtained polymer, the acrylamide derivative Bresidues to which a neoglycopeptide was bound had the followingstructural formula, wherein Ac is an acetyl group, and were contained ina proportion of 9 mol %.

REFERENCE EXAMPLE 20 Synthesis of Acrylamide Derivative A/AcrylamideCopolymer (Copolymerization Ratio=1:10, Sugar Chain-Having Polymer P)

A reaction was carried out in the same manner as in Example 15 exceptfor using 35.5 mg of acrylamide in place of 14.4 mg of acrylic acid and21.3 mg of acrylamide, to afford 90 mg of the target compound (sugarchain-having polymer P). In the obtained polymer, the acrylamidederivative A residues to which a glycopeptide was bonded were containedin a proportion of 9 mol %.

REFERENCE EXAMPLE 21 Synthesis of Acrylamide Derivative B/AcrylamideCopolymer (Copolymerization Ratio=1:10, Sugar Chain-Having Polymer Q)

A reaction was carried out in the same manner as in Example 15 exceptfor using 35.5 mg of acrylamide in place of 14.4 mg of acrylic acid and21.3 mg of acrylamide, and using 61 mg of acrylamide derivative Bobtained in Reference Example 19 in place of 60 mg of acrylamidederivative A obtained in Reference Example 18, to afford 91 mg of thetarget compound (sugar chain-having polymer Q).

EXAMPLE 17 Galactose Transfer to Sugar Chain-Having Polymer N byImmobilized β1,4-Galactosyltransferase, and Release of Glycopeptide fromSugar Chain-Having Polymer N by α-Chymotrypsin

One milliliter of the immobilized β1,4-galactose transferase obtained inReference Example 9 and 38 mg of sugar chain-having polymer N obtainedin Example 15 were incubated in 2 ml of 50 mM HEPES buffer (pH 7.0)containing 50 mM UDP-Gal and 10 mM manganese chloride with shaking at37° C. for 48 hours. After the incubation, the reaction mixture wassubjected to column chromatography on Sephadex G-25 (Pharmacia) (eluant:10 mM ammonium acetate). The void fraction, the fraction containing aproduct, was then lyophilized to afford 36 mg of a product. A solutionof the product (10 mg) and 0.3 mg of α-chymotrypsin (0.3 mg) in 80 mMtris-hydrochloric acid buffer solution (2 ml, pH 7.8, containing 0.1 Mcalcium chloride) was incubated at 40° C. for 24 hours to release aglycopeptide whose sugar chain had been elongated, from the sugarchain-having polymer. The reaction mixture was subjected to columnchromatography on Sephadex G-25 (Pharmacia) (eluant: 10 mM aceticammonium). The fraction containing a product was lyophilized to afford 6mg of a glycopeptide whose sugar chain had been elongated. The H-NMRspectrum of the obtained glycopeptide was measured, showing that theglycopeptide had the following structural formula, wherein Ac is anacetyl group, and that the galactose transfer reaction had proceededquantitatively.

EXAMPLE 18 Galactose Transfer to Sugar Chain-Having Polymer O byImmobilized β1,4-Galactosyltransferase, and Release of Neoglycopeptidefrom Sugar Chain-Having Polymer O by α-Chymotrypsin

A reaction was carried out in the same manner as in Example 17 exceptfor using 39 mg of sugar chain-having polymer 0 obtained in Example 16in place of 38 mg of sugar chain-having polymer N obtained in Example15, to afford 37 mg of a product. Using 10 mg of the product, theprocedure of Example 17 was followed to release a neoglycopeptide whosesugar chain had been elongated, from the sugar chain-having polymer. TheH-NMR spectrum of the obtained neoglycopeptide was measured, showingthat the neoglycopeptide had the following structural formula, whereinAc is an acetyl group, and that the galactose transfer reaction hadproceeded quantitatively.

REFERENCE EXAMPLE 22 Galactose Transfer to Sugar Chain-Having Polymer Pby Immobilized β1,4-galactosyltransferase

A reaction was carried out in the same manner as in Example 17 exceptfor using 38 mg of sugar chain-having polymer P obtained in ReferenceExample 20 in place of 38 mg of sugar chain-having polymer N obtained inExample 15, to afford 36 mg of a product. The H-NMR spectrum of theobtained product was measured, showing that the product containedtransferred galactose. In the polymer containing transferred galactose,the acrylamide derivative A residues to which a glycopeptide was boundhad the following structural formula, wherein Ac is an acetyl group.

Reference Example 23 Galactose Transfer to Sugar Chain-Having Polymer Qby Immobilized β1,4-galactosyltransferase

A reaction was carried out in the same manner as in Example 17 exceptfor using 39 mg of sugar chain-having polymer Q obtained in ReferenceExample 21 in place of 38 mg of sugar chain-having polymer N obtained inExample 15, to afford 37 mg of a product. The H-NMR spectrum of theobtained product was measured, showing that the product containedtransferred galactose. In the polymer containing transferred galactose,the acrylamide derivative B residues to which a glycopeptide was bound,had the following structural formula, wherein Ac is an acetyl group.

EXAMPLE 19 Sialic Acid Transfer to Sugar Chain-Having Polymers N and Pby Immobilized α2,3-sialyltransferase

One milliliter of the immobilized α2,3-sialyltransferase obtained inReference Example 10, and either 21 mg of sugar chain-having polymer Ncontaining transferred galactose obtained in Example 17, or 21 mg ofsugar chain-having polymer P containing transferred galactose obtainedin Reference Example 22, were incubated in 2 ml of 50 mM sodiumcacodylate buffer (pH 7.0) containing 50 mM CMP-NeuAc, 10 mM manganesechloride and 0.1% Triton CF-54 at 37° C. for 18 hours. After theincubation, the reaction mixture was subjected to column chromatographyon Sephadex G-25 (Pharmacia) (eluant: 10 mM ammonium acetate). Thefraction containing a product was lyophilized. 17 mg each of twoproducts was thereby obtained. Using 10 mg each of the obtainedproducts, the procedure of Example 17 was followed to releaseglycopeptides whose sugar chain had been elongated, from the sugarchain-having polymers. The H-NMR spectra of the released glycopeptideswere measured, and based on the spectra, the conversions of the sialicacid transfer reactions were compared. The results were that theconversion of sugar chain-having polymer N was about 80%, whereas thatof sugar chain-having polymer P was about 50%, demonstrating that sugarchain-having polymer N had superior reactivity.

EXAMPLE 20 Sialic Acid Transfer to Sugar Chain-Having Polymers 0 and QUsing Immobilized α2,3-sialyltransferase

A reaction was carried out in the same manner as in Example 19 exceptfor using 22 mg of sugar chain-having polymer 0 containing transferredgalactose obtained in Example 18, or 22 mg of sugar chain-having polymerQ containing transferred galactose obtained in Reference Example 23, inplace of 21 mg of sugar chain-having polymer N containing transferredgalactose obtained in Example 17, or 21 mg of sugar chain-having polymerP containing transferred galactose obtained in Reference Example 20, toafford 18 mg each of two products. Using 10 mg each of the obtainedproducts, the procedure of Example 17 was followed to releaseneoglycopeptides whose sugar chain had been elongated, from the sugarchain-having polymers. The H-NMR spectra of the releasedneoglycopeptides were measured, and based on the spectra, theconversions of the sialic acid transfer reactions were compared. Theresults were that the conversion of sugar chain-having polymer O wasabout 80%, whereas that of sugar chain-having polymer Q was about 60%,demonstrating that sugar chain-having polymer O had superior reactivity.

EXAMPLE 21 Sialic Acid Transfer to Sugar Chain-Having Polymer N byImmobilized α2,3-sialyltransferase, and Release of Glycopeptide fromSugar Chain-Having Polymer N by α-chymotrypsin

A reaction was carried out in the same manner as in Example 19 exceptthat the reaction was performed for 24 hours using 100 mM CMP-NeuAc and1.5 ml of the immobilized α2,3-sialyltransferase obtained in ReferenceExample 10 in place of 50 mM CMP-NeuAc and 1.0 ml of the immobilizedα2,3-sialyltransferase obtained in Reference Example 10, to afford 17 mgof a product. The procedure of Example 17 was followed to release aglycopeptide whose sugar chain had been elongated, from the sugarchain-having polymer, and the H-NMR spectrum of the obtainedglycopeptide was measured, showing that the glycopeptide had thefollowing structural formula, wherein Ac is an acetyl group, and thatthe sialic acid transfer reaction proceeded nearly quantitatively.

REFERENCE EXAMPLE 24 Synthesis ofN-(benzyloxycarbonylphenylalanyl)-6-amino-1-hexanol

To a solution of N-benzyloxycarbonylphenylalanine (11.96 g) and6-amino-1-hexanol (5.2 g) in a mixed solvent (40 ml) ofbenzene:ethanol=1:1 was addedN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (hereinafter EEDQ) (9.9g), followed by stirring at room temperature for 24 hours. After thereaction, the reaction mixture was evaporated, and the residue wasrecrystallized from benzene to give the target compound (13.6 g).N-(benzyloxycarbonylphenylalanyl)-6-amino-1-hexanol had the followingstructural formula.

REFERENCE EXAMPLE 25 Synthesis ofN-(benzyloxycarbonylphenylalanyl)-6-aminohexyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside

To a solution of the2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline(2.96 g) obtained in Reference Example 1 and theN-(benzyloxycarbonylphenylalanyl)-6-amino-1-hexanol (7.17 g) obtained inReference Example 24 in dichloroethane (35 ml) was addedD-camphor-10-sulfonic acid (hereinafter CSA) to be pH 2-3 at 70° C.After stirring for 30 minutes the reaction mixture was cooled to roomtemperature, diluted with chloroform and washed twice with saturatedaqueous sodium hydrogencarbonate solution. The organic layer was driedover anhydrous magnesium sulfate overnight. The magnesium sulfate wasfiltered off through celite, and the filtrate was concentrated underreduced pressure and subjected to silica gel chromatography (eluant;chloroform). The eluate was evaporated to give the target compound (2.37g). N-(benzyloxycarbonylphenylalanyl)-6-aminohexyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside had thefollowing structural formula, wherein Ac is an acetyl group.

REFERENCE EXAMPLE 26 Synthesis ofN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside

To a solution of theN-(benzyloxycarbonylphenylalanyl)-6-aminohexyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(1.5 g) obtained in Reference Example 25 in methanol (40 ml) was added10% palladium-carbon (150 mg). The reaction mixture was stirred underhydrogen atmosphere at 50° C. for 2 hours. The catalyst was filteredoff, and the filtrate was concentrated under reduced pressure. To asolution of the residue and the 6-acryloylaminocaproic acid (0.42 g)obtained in Reference Example 3 in mixed solvent of ethanol:benzene=1:1was added EEDQ (0.55 g), followed by stirring at room temperature for 24hours. The reaction mixture was concentrated under reduced pressure, andthe residue was recrystallized from ethanol to give the target compound(1.2 g).N-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosidehad the following structural formula, wherein Ac is an acetyl group.

REFERENCE EXAMPLE 27 Synthesis ofN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-2-deoxy-β-D-glucopyranoside

To a solution of theN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(590 mg) obtained in Reference Example 26 in mixed solvent ofTHF:methanol=1:1 (20 ml) was added sodium methoxide (16.9 mg), followedby stirring at room temperature for 24 hours. A cation exchange resin,Dowex 50WX-8 (H⁺) (Dow Chemical Co.), was added to be pH 7. The ionexchange resin was filtered off, and the filtrate was concentrated underreduced pressure. The residue was recrystallized from mixed solvent ofethanol:benzene=1:1 to give the target compound (413 mg).N-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-2-deoxy-β-D-glucopyranosidehad the following structural formula, wherein Ac is an acetyl group.

EXAMPLE 22 Synthesis ofN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-2-deoxy-β-D-glucopyranoside/acrylicAcid/Acrylamide Copolymer (Copolymerization Ratio=1:2:7, SugarChain-Having Polymer R)

To a solution of theN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-2-deoxy-β-D-glucopyranoside(79.3 mg) obtained in Reference Example 27, acrylic acid (18.0 mg) andacrylamide (62.2 mg) in mixed solvent of DMSO:water=3:1 (1 ml) wereadded TEMED (11.6 μl) and ammonium peroxodisulfate (8.6 mg), andcopolymerization was carried out at room temperature for 24 hours. Thereaction mixture was concentrated under reduced pressure, DMSO wasdistilled off, and the residue was subjected to column chromatography onSephadex G-25 (Pharmacia) (eluant; 10 mM ammonium acetate). The targetcompound fraction was lyophilized to afford the target compound (160mg). TheN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-2-deoxy-β-D-glucopyranosideresidues in the obtained polymer had the following structural formula.

REFERENCE EXAMPLE 28 Synthesis ofN-(6′-acryloylaminocaproyl)phenylalanyl-6-aminohexyl-2-acetamide-2-deoxy-β-D-glucopyranoside/acrylamideCopolymer (Copolymerization Ratio=1:9, Sugar Chain-Having Polymer S)

A reaction was carried out in the same manner as in Example 22, exceptfor using 80.0 mg of acrylamide in place of 18.0 mg of acrylic acid and62.2 mg of acrylamide, to afford 160 mg of the target compound.

EXAMPLE 23 Galactose Transfer to Sugar Chain-Having Polymer R byImmobilized β1,4-galactosyltransferase

0.5 ml of the immobilized β1,4-galactosyltransferase obtained inReference Example 9 and 13.5 mg of sugar chain-having polymer R obtainedin Example 22 were incubated with shaking in 50 mM HEPES buffer (1.0 ml,pH 7.0) containing 20 mM UDP-Gal and 10 mM manganese chloride at 37° C.for 24 hours. After the incubation, the immobilizedβ1,4-galactosyltransferase was removed by centrifugation, and theresulting reaction mixture was subjected to column chromatography onSephadex G-25 (Pharmacia) (eluant: 50 mM ammonium formate). The fractioncontaining a product was lyophilized to afford a product (11 mg). TheH-NMR spectrum of the obtained product was measured, showing thatgalactose had been quantitatively transferred.

REFERENCE EXAMPLE 29 Galactose Transfer to Sugar Chain-Having Polymer Sby Immobilized β1,4-galactosyltransferase

A reaction was carried out in the same manner as in Example 23 exceptfor using 13.5 mg of sugar chain-having polymer S obtained in Example 28in place of 13.5 mg of sugar chain-having polymer R obtained inReference Example 22, to afford 11 mg of a product. The H-NMR spectrumof the obtained product was measured, showing that galactose had beenquantitatively transferred.

REFERENCE EXAMPLE 30 Preparation of Immobilized β2,6-sialyltransferase

The procedure of Reference Example 10 was followed except for using 1 Uof rat liver-derived α2,6-sialyltransferase in place of 1 U ofα2,3-sialyltransferase, to prepare the target compound, which was thenstored at 4° C. The obtained immobilized enzyme had an activity of 120mU/ml.

EXAMPLE 24 Sialic Acid Transfer to Sugar Chain-Having Polymers R and Sby Immobilized α2,6-sialyltransferase

0.5 ml of the immobilized α2,6-sialyltransferase obtained in ReferenceExample 30, and either 7.5 mg of sugar chain-having polymer R containingtransferred galactose obtained in Example 23, or 7.5 mg of sugarchain-having polymer S containing transferred galactose obtained inReference Example 29, were incubated with shaking in 50 mM sodiumcacodylate buffer (1 ml, pH 7.4) containing 25 mM CMP-NeuAc and 10 mMmanganese chloride at 37° C. for 24 hours. The procedure of Example 23was followed to afford 6 mg each of two products. The H-NMR spectra ofthe obtained products were measured and the conversions of the sialicacid transfer reactions were compared. The results were that thereaction proceeded quantitatively in sugar chain-having polymer R,whereas only 70% of the reaction proceeded in sugar chain-having polymerS.

EXAMPLE 25 Release of Glycoconjugate from Sugar Chain-Having Polymer Rby α-chymotrypsin

Five milligrams of sugar chain-having polymer R containing transferredsialic acid obtained in Example 24 and 0.6 mg of α-chymotrypsin wereincubated in 80 mM tris-hydrochloric acid buffer (2 ml, pH 7.8,containing 0.1 M calcium chloride) at 40° C. for 24 hours. The reactionmixture was subjected to column chromatography on Sephadex G-25(Pharmacia) (eluant: 50 mM ammonium formate). The fraction containing aproduct was lyophilized to afford a product (2 mg). The H-NMR spectrumof the obtained product was measured, showing that the product had thefollowing structural formula.

REFERENCE EXAMPLE 31 Synthesis of N-benzyloxycarbonylserine octylamide

To a solution of N-benzyloxycarbonylserine (12 g) in mixed solvent ofethanol:benzene=1:1 (120 ml) were added EEDQ (13.6 g) and octylamine(11.1 ml), followed by stirring at room temperature overnight. Thereaction mixture was concentrated under reduced pressure andrecrystallized from toluene to give the target compound (12.64 g).N-benzyloxycarbonylserine octylamide had the following structuralformula.

REFERENCE EXAMPLE 32 Synthesis ofO-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosyl-N-benzyloxycarbonylserineOctylamide

To a solution of the N-benzyloxycarbonylserine octylamide (4.0 g)obtained in Reference Example 31 and dried thoroughly preliminary indichloroethane (80 ml) were added activated molecular sieves 4 Å (8.0 g)and 2,3,6,2′,3′,4′,6′-hepta-O-acetyllactosyl bromide (12.0 g). Silvertrifluoromethanesulfonate (4.40 g) was added while chilling in icewater, and the resulting mixture was stirred overnight under a stream ofnitrogen while gradually being allowed to room temperature. The reactionmixture was filtered through celite, and the filtrate was washed twicewith saturated saline solution and then dried over anhydrous magnesiumsulfate. After the drying, the magnesium sulfate was filtered off, andthe filtrate was concentrated under reduced pressure and thereaftersubjected to silica gel column chromatography (eluant: toluene/ethylacetate=5/1). The eluate was evaporated to give the target compound(5.32 g).O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosyl-N-benzyloxycarbonylserineoctylamide had the following structural formula, wherein Ac is an acetylgroup.

REFERENCE EXAMPLE 33 Synthesis ofO-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosylserine Octylamide

4.0 g of theO-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosyl-N-benzyloxycarbonylserineoctylamide obtained in Reference Example 32 was dissolved in methanol(60 ml), and catalytic reduction was carried out under hydrogenatomosphere at room temperature in the presence of 5% palladium-carbonas catalyst. After the reaction, the catalyst was filtered off, and thereaction mixture was evaporated to afford the target compound (3.42 g).O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosylserine octylamide had thefollowing structural formula, wherein Ac is an acetyl group.

REFERENCE EXAMPLE 34 Synthesis ofO-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosyl-N-(6-acryloylamino)caproylserineOctylamide

To a solution of the 6-acryloylaminocaproic acid (278 mg) obtained inReference Example 3 and EEDQ (371 mg) in mixed solvent ofethanol:benzene=1:1 (40 ml) was added0-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosylserine octylamide (1.14 g)obtained in Reference Example 33, followed by stirring at roomtemperature overnight. The reaction mixture was concentrated underreduced pressure and subjected to silica gel chromatography (eluant:chloroform/methanol=100/1). The fraction containing the target compoundwas evaporated to give the target compound (1.06 g).O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosyl-N-(6-acryloylamino)caproylserineoctylamide had the following structural formula, wherein Ac is an acetylgroup.

REFERENCE EXAMPLE 35 Synthesis ofO-lactosyl-N-(6-acryloylamino)caproylserine Octylamide

Four hundred milligrams ofO-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl)lactosyl-N-(6-acryloylamino)caproylserineoctylamide obtained in Reference Example 34 was dissolved in anappropriate amount of mixed solvent of tetrahydrofuran:methanol=1:1, andsodium methoxide (8.49 mg) was added, followed by stirring at roomtemperature for 2 hours. The solution was neutralized by adding a cationexchange resin, Dowex 50WX-8 (H⁺) (Dow Chemical Co.). The ion exchangeresin was filtered off, and the filtrate was concentrated under reducedpressure and recrystallized from ethanol to give the target compound(270 mg). O-lactosyl-N-(6-acryloylamino)caproylserine octylamide had thefollowing structural formula.

EXAMPLE 26 Synthesis of O-lactosyl-N-(6-acryloylamino)caproylserineoctylamide/acrylic acid/acrylamide Copolymer (CopolymerizationRatio=1:2:7, Sugar Chain-Having Polymer T)

To a solution of the O-lactosyl-N-(6-acryloylamino)caproylserineoctylamide (70.8 mg) obtained in Reference Example 35, acrylic acid(14.4 mg) and acrylamide ('49.8 mg) in appropriate amount of mixedsolvent of DMSO:water=1:1 were added TEMED (12 μl) and ammoniumperoxodisulfate (7.67 mg), followed by polymerization at 50° C.overnight. The target compound was purified by gel filtrationchromatography on Sephadex G-25 (Pharmacia) column pre-equilibrated withdistilled water. The fraction containing the target compound waslyophilized to afford the target compound (117 mg). TheO-lactosyl-N-(6-acryloylamino)caproylserine octylamide residues in theobtained polymer had the following structural formula.

REFERENCE EXAMPLE 36 Synthesis ofO-lactosyl-N-(6-acryloylamino)caproylserine octylamide/acrylamideCopolymer (Copolymerization Ratio=1:9, Sugar Chain-Having Polymer U)

A reaction was carried out in the same manner as in Example 26, exceptfor using 64.0 mg of acrylamide in place of 14.4 mg of acrylic acid and49.8 mg of acrylamide, to afford the target compound (105 mg).

EXAMPLE 27 Sialic Acid Transfer to Sugar Chain-Having Polymers T and Uby Immobilized α2,3-sialyltransferase

0.5 ml of the immobilized α2,3-sialyltransferase obtained in ReferenceExample 10, and either 13.5 mg of sugar chain-having polymer T obtainedin Example 28 or 16.4 mg of sugar chain-having polymer U were incubatedin 50 mM sodium cacodylate buffer (1.0 ml, pH 7.0) containing 50 mMCMP-NeuAc and 10 mM manganese chloride at 30° C. for 24 hours. After theincubation, the immobilized α2,3-sialyltransferase was removed bycentrifugation, and the obtained reaction mixture was subjected tocolumn chromatography on Sephadex G-25 (Pharmacia) (eluant: 50 mMammonium formate), the eluate was then lyophilized. Thus, two productswere obtained in amounts of 14 mg and 12 mg, respectively. The H-NMRspectra of the obtained products were measured, and the conversions ofthe sialic acid transfer reactions were compared. The results were thatthe conversion of sugar chain-having polymer T was about 90%, whereasthat of sugar chain-having polymer U was about 70%. Theo-lactosyl-N-(6-acryloylamino)caproylserine octylamide residues in thepolymer containing transferred sialic acid had the following structuralformula.

EXAMPLE 28 Comparison of Recoveries of Sugar Chain-Having Polymers byGel Filtration Chromatography

A solution of either sugar chain-having polymer S or T (10 mg)containing transferred sialic acid obtained in Example 27 in 50 mMsodium cacodylate buffer (2.0 ml, pH 7.0) containing 50 mM CMP-NeuAc and10 mM manganese chloride was subjected to column chromatography onSephadex G-25 (Pharmacia) (eluant: 50 mM ammonium formate), andrecovered the sugar chain-having polymer. The recoveries of the twopolymers were compared. The results were that the recovery of sugarchain-having polymer S was 87%, whereas that of sugar chain-havingpolymer T was 77%, showing that the recovery of sugar chain-havingpolymer S was superior.

EXAMPLE 29 Sugar Chain Transfer from Sugar Chain-Having Polymer toN-stearoylsphingosine Using Ceramide Glycanase

Leech-derived ceramide glycanase (0.01 U) was added to 50 mM citric acidbuffer (1.0 ml, pH 6.0) containing 10 mg of the sugar chain-havingpolymer containing transferred sialic acid obtained in Example 27, 25 mgof N-stearoylsphingosine and 20 μl of triton CF-54, followed byincubating at 37° C. for 17 hours. After the incubation, a product wasseparated by column chromatography on Sephadex LH-20 (Pharmacia) columnequilibrated with chloroform:methanol:water=60:30:5. The fractioncontaining the product was evaporated to afford the product (6 mg). HPLCanalysis revealed that the product was1-O-(N-acetylneuraminyl-α-(2→3))lactosyl-N-stearoylsphingosine.1-O-(N-acetylneuraminyl-α-(2→3))lactosyl-N-stearoylsphingosine had thefollowing structural formula, wherein Ac is an acetyl group.

INDUSTRIAL APPLICABILITY

Use of the sugar chain-having polymer of the present invention forglycoconjugate synthesis enables easy and efficient synthesis of variousglycoconjugates, for example, oligosaccharides, glycopeptides,glycolipids and glycosides. The obtained glycoconjugates have variousphysiological activities, and are therefore expected to findapplications in medicines and other fields.

1. A water-soluble polymer compound having sugar chain(s) comprising amonosaccharide or an oligosaccharide residue bound to side chain(s) of awater-soluble polymer through a linker containing a selectivelycleavable bond, the water-soluble polymer containing 20 to 80 mol % of(meth)acrylic acid residue, and the linker being bonded to a repeatingunit other than (meth)acrylic acid residue.
 2. A compound according toclaim 1, wherein amino acid or peptide residues bound to amonosaccharide or an oligosaccharide residue are linked to side chain(s)of the water-soluble polymer through a linker containing a selectivelycleavable bond, the water-soluble polymer containing 20 to 80 mol % of(meth)acrylic acid residue, and the linker being bound to a repeatingunit other than (meth)acrylic acid residue.
 3. A compound according toclaim 1, wherein the water-soluble polymer is a copolymer comprising 20to 80 mol % of (meth)acrylic acid and 80 to 20 mol % of one or morevinyl monomers selected from the group consisting of acrylamidederivatives, methacrylamide derivatives, acrylic esters, methacrylicesters, styrene derivatives and fatty-acid vinyl esters.
 4. A compoundaccording to claim 1, wherein the selectively cleavable bond containedin the linker can be cleaved by hydrogenolysis or by oxidation using2,3-dichloro-5,6-dicyanobenzoquinone.
 5. A compound according to claim1, wherein the linker is a group represented by General Formula (I),

wherein R¹ is a monosaccharide or an oligosaccharide residue, R² is abivalent linking group with a length equivalent to 4 to 20 methylenegroups, and X is O, S, or NH.
 6. A compound according to claim 5,wherein R¹ is an N-acetylglucosamine residue, a glucose residue or alactose residue.
 7. A compound according to claim 5, wherein R² is apentylene group.
 8. A compound according to claim 1, wherein the linkeris a group represented by General Formula (II),

wherein R³ is a monosaccharide or an oligosaccharide residue, R⁴ is aC₆₋₂₀ alkyl or alkenyl group, R⁵ is a bivalent linking group with alength equivalent to 5 to 19 methylene groups, and Y is O, S, or NH. 9.A compound according to claim 8, wherein R³ is a glucose or lactoseresidue.
 10. A compound according to claim 1, wherein the linker is agroup represented by General Formula (III),

wherein R⁶ is a monosaccharide or an oligosaccharide residue, R⁷ is abivalent linking group with a length equivalent to 2 to 20 methylenegroups, R⁸ is a bivalent linking group with a length equivalent to 5 to19 methylene groups, and Z and W are each independently O, S, or NH. 11.A compound according to claim 10, wherein R⁶ is an N-acetylglucosamineresidue.
 12. A compound according to claim 2, wherein the peptideresidue consists of 2 to 30 amino acid residues.
 13. A compoundaccording to claim 1, wherein the selectively cleavable bond containedin the linker can be cleaved by an appropriate hydrolase.
 14. A compoundaccording to claim 13, wherein the appropriate hydrolase is ceramideglycanase or α-chymotrypsin.
 15. A compound according to claim 13,wherein the appropriate hydrolase is a protease that does not have acleavage site in an amino acid or peptide residue to which amonosaccharide or an oligosaccharide residue is bound.
 16. A compoundaccording to claim 15, wherein the linker containing a selectivelycleavable bond that is linked to an amino acid or a peptide residuebound to a monosaccharide or an oligosaccharide residue is a grouprepresented by General Formula (IV),—R⁹-R¹⁰—  (IV) wherein R⁹ is a bivalent linking group with a lengthequivalent to 1 to 20 methylene groups and is linked to thewater-soluble polymer compound, and R¹⁰ is an amino acid or a peptideresidue containing a cleavable site by an appropriate protease and isbound to a monosaccharide or an oligosaccharide residue, and that themonosaccharide or oligosaccharide residue is bound to a side chainfunctional group of Asn, Asp, Cys, Gln, Glu, Lys, Ser, Thr or Tyrresidue, or to a side chain functional group of the amino acid residuein a peptide residue directly or through a bivalent linking group via aglycosidic bond.
 17. A compound according to claim 16, wherein R⁹ is agroup represented by General Formula (V),-A-(CH₂)_(n)—CO—  (V) wherein A is O, CH₂, C═O, or NH, the group islinked to a side chain of the water-soluble polymer through A, and n isan integer from 1 to
 18. 18. A compound according to claim 16, whereinthe bivalent linking group bound to the side chain functional group is agroup with a length equivalent to 1 to 20 methylene groups.
 19. Acompound according to claim 16, wherein the bivalent linking grouplinked to the side chain functional group is a group represented byGeneral Formula (VI),—B—(CH₂)_(n)—O—  (VI) wherein B is O, NH, or C═O, the group is linked tothe side chain functional group of an amino acid residue through B, andn is an integer from 1 to
 18. 20. A water-soluble polymer primer forglycoconjugate synthesis comprising a water-soluble polymer compoundhaving sugar chain(s) according to claim
 1. 21. A method for producing awater-soluble polymer compound having sugar chain(s) comprising a stepof copolymerization of (meth)acrylic acid, a (meth)acrylamide derivativerepresented by General Formula (VII),

wherein R¹¹ is a monosaccharide or an oligosaccharide residue, and R¹²is a bivalent linking group with a length equivalent to 4 to 20methylene groups, and at least one vinyl monomer in such a manner thatthe proportion of the (meth)acrylic acid in the total vinyl copolymersis 20 to 80 mol %.
 22. A method according to claim 21, wherein R¹¹ is anN-acetylglucosamine residue, a glucose residue, or a lactose residue.23. A method according to claim 21, wherein R¹² is a pentylene group.24. A method according to claim 21, wherein the vinyl monomer is atleast one monomer selected from the group consisting of acrylamidederivatives, methacrylamide derivatives, acrylic esters, methacrylicacid esters, styrene derivatives, and fatty acid vinyl esters.
 25. Amethod for producing a glycoconjugate comprising the steps of:transferring a sugar residue from a sugar nucleotide to a polymercompound by contacting a water-soluble polymer compound having sugarchain(s) of claim 1 with a glycosyltransferase in the presence of asugar nucleotide, elongating the sugar chain by repeating step 1 two ormore times if necessary, removing by-product nucleotides or unreactedsugar nucleotides if necessary, and after repeating steps 1 to 3 two ormore times, releasing the resultant glycoconjugate sugar chain from thewater-soluble polymer compound which binds the sugar chain elongated bythe transfer of the plurality of sugar residues.
 26. A method forproducing a glycoconjugate comprising the steps of: transferring a sugarresidue from a sugar nucleotide to a water-soluble polymer compound bythe action of a glycosyltransferase to the water-soluble polymercompound having sugar chain(s) of claim 8 in the presence of a sugarnucleotide, elongating the sugar chain by repeating step 1 two or moretimes if necessary, removing by-product nucleotides or unreacted sugarnucleotides if necessary and after repeating steps 1 to 3 two or moretimes, transferring the resultant oligosaccharide elongated by transferof the plurality of sugar residues from the water-soluble polymercompound to ceramide by the action of ceramide glycanase in the presenceof ceramide.